阅读说明：本技术 一种筛选牛高原低氧适应基因aldoc和功能性分子标记的方法及其应用 (Method for screening bovine plateau hypoxia adaptive gene ALDOC and functional molecular marker and application thereof ) 是由 黄金明 魏晓超 刘文浩 杨春红 王金鹏 鞠志花 王秀革 姜强 张亚冉 高亚平 王 于 2020-11-27 设计创作，主要内容包括：本发明提供一种筛选牛高原低氧适应基因ALDOC和功能性分子标记的方法及其应用,属于动物分子育种技术领域。本发明从牛基因组选择和遗传适应等角度,选择分布于高海拔和低海拔的牛品种,通过采用SNP芯片,整合FLK、hapFLK和XPEHH三种基因组选择信号分析方法,比较高低海拔牛之间和牛品种内的基因组选择信号,通过组合筛选策略和生物信息学分析鉴定出适应高原低氧极端环境的候选基因,进一步通过选择信号验证和重测序数据分析挖掘出潜在的功能性分子标记,并建立相应检测方法。本发明为高原低氧特色牛品种的培育等分子育种提供科学依据和简单易行的检测技术；同时对地方牛品种遗传资源的保护、评价和利用具重要意义和价值。(The invention provides a method for screening a bovine plateau hypoxia adaptive gene ALDOC and a functional molecular marker and application thereof, belonging to the technical field of animal molecular breeding. The method selects cattle varieties distributed at high altitude and low altitude from the aspects of cattle genome selection, genetic adaptation and the like, integrates three genome selection signal analysis methods of FLK, hapFLK and XPEHH by adopting an SNP chip, compares the genome selection signals between cattle at high altitude and in the cattle varieties, identifies candidate genes adapting to high-altitude hypoxia extreme environments by combined screening strategies and bioinformatics analysis, further excavates potential functional molecular markers by selecting signal verification and re-sequencing data analysis, and establishes a corresponding detection method. The invention provides scientific basis and simple and easy detection technology for molecular breeding such as breeding of plateau hypoxia special cattle variety; meanwhile, the method has important significance and value for the protection, evaluation and utilization of local cattle variety genetic resources.)

1. A method for screening a bovine plateau hypoxia adaptive gene and a functional molecular marker, the method at least comprising:

DNA samples of different altitude cattle varieties are detected and analyzed based on the SNP chip;

the method is characterized in that analysis is carried out based on FLK, hapFLK and XPEHH genome selection signal analysis methods, single nucleotide polymorphic SNPs and candidate genes subjected to significant positive selection are screened, and plateau hypoxia adaptive candidate genes and SNP molecular markers are screened by integrating gene function annotation.

2. The method of claim 1, wherein the method for screening the bovine plateau hypoxia adaptive gene and the functional molecular marker comprises:

s1, collecting and DNA extracting samples of cattle varieties at different altitudes;

s2, detecting and analyzing the SNP chip;

s3, analyzing FLK and hapFLLK genome selection signals;

s4, XPEHH genome selection signal analysis;

s5, identifying a positively selected genetic variation and a differentially selected region;

s6, screening candidate genes based on a screening strategy of the candidate genes;

s7, verifying a positive selection signal of the difference selection area;

s8, identifying potential functional variation in the positive selection gene by using the remeasured data;

wherein, the steps S3 and S4 are not in sequence.

3. The method as claimed in claim 2, wherein the step S1 includes the following steps:

s1.1, selecting common cattle and tumor cattle varieties at different altitudes, and mixed varieties of the common cattle and the tumor cattle;

s1.2, blood of a cow is collected, and DNA in blood tissues is extracted.

4. The method as claimed in claim 2, wherein the step S2 includes the following steps:

s2.1, analyzing the sample by using the SNP chip, and genotyping;

s2.2, filtering the SNP data, and further analyzing the remaining SNPs meeting the requirements;

s2.3 haplotype was constructed for each chromosome.

5. The method as claimed in claim 2, wherein the step S3 includes the following steps:

s3.1, respectively comparing selection signal values corresponding to SNPs of higher altitude and lower altitude groups based on an FLK and hapFLK whole genome selection signal analysis method, and acquiring difference selection signals of the SNPs between the two groups;

s3.2, respectively operating FLK and hapFLK programs by using genotype data to obtain selection signals of SNPs in the variety, and taking Nelore as a distant population; in the FLK analysis, Nelore is defined as the distant population, and K10 and nti 20;

s3.3, constructing an evolutionary tree of the whole genome and a local genome region for the selected region by using hapFLK results and Python and R scripts;

s3.4 p-value of hapFLK values were calculated by fitting a standard normal distribution of the whole genome in R.

6. The method as claimed in claim 2, wherein the step S4 includes the following steps:

s4.1, estimating XPEHH values between high-altitude and low-altitude cattle varieties, and defining the relation between physical distance and genetic distance by using 1Mb ≈ 1cM in cattle genome;

s4.2 construction of haplotypes based on ReqDel-FAST.

7. The method as claimed in claim 2, wherein the step S5 includes the following steps:

respectively screening SNPs with positive selection signal values of 0.1% in the front based on FLK, hapFLK and XPEHH selection signal analysis results; or the like, or, alternatively,

the specific method of step S6 includes:

s6.1, defining 50Kbp of the upstream and downstream SNPs obtained by identification as selected areas, and connecting overlapping areas in series to obtain a differential selection area;

s6.2, annotating a reference gene in the difference selection area or overlapped with the area by using the UCSC online website;

s6.3, screening out the FLK, hapFLK and XPEHH selection signals which are compared among high and low altitude groups and selecting the difference selection areas which are 10 th from the top in rank, respectively identifying candidate genes and carrying out functional annotation.

8. The method as claimed in claim 2, wherein the step S7 includes the following steps:

s7.1, respectively constructing a whole genome and a local selection region evolutionary tree based on FLK and hapFLK analysis results, and analyzing the selection condition of the candidate gene in each cattle variety;

s7.2 the results of the comparisons between groups were further validated based on the results of the in-breed FLK and hapFLLK selection signal analysis.

9. The method as claimed in claim 2, wherein the step S8 includes the following steps:

s8.1, acquiring double-end Illumina re-sequencing data, and comparing the acquired data with reference genomes;

s8.2, filtering to obtain a high-quality mapping sequence, and detecting genetic variation from the high-quality mapping sequence;

s8.3, extracting genetic variation from a specific gene region, wherein the extracted regions are an upstream region and a gene region of a candidate gene;

s8.5, respectively calculating the genotype and the allele frequency of each genetic variation;

s8.6, determining potential SNP sites of functional variation through functional annotation;

preferably, the bovine plateau hypoxia adaptive gene is ALDOC gene;

preferably, the bovine plateau hypoxia adaptive molecular marker further comprises a single nucleotide polymorphism site located in the ALDOC gene, including but not limited to g.20588331T > G (rs133198943A > C); g.20590099G > A (c.288C > T, aa96F > F); g, 20590664T > C; g.20591903C > A (g.20591903G > T).

10. Use of the method of any one of claims 1-9 for screening bovine individuals (populations, lines or breeds) suitable for high altitude hypoxic survival;

preferably, the application mode specifically comprises:

extracting blood DNA of different cattle individuals;

identifying the genotype of the marker by a CAS-PCR-RFLP method by using the kit for detecting the molecular marker, and identifying individuals with the plateau hypoxia adaptation specific molecular marker;

further preferably, the molecular marker is a single nucleotide polymorphic site g.20591903G > T;

in this case, the kit comprises at least the following primers and BstUI restriction enzyme;

F:5‘-GTAATTGTTTACGGTGACGC-3’(SEQ ID NO.7)；

R:5‘-GGCCTTGTTCTGATTCCTGC-3’(SEQ ID NO.8)。

Technical Field

The invention belongs to the technical field of animal molecular breeding, and particularly relates to a method for screening a bovine plateau hypoxia adaptive gene ALDOC and a functional molecular marker and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Cattle are ruminants that live in different environments with the ability to efficiently convert low quality feed into high energy fats, meat and milk (Elsik et al, 2009). Under the action of rumen microorganisms, cattle digest roughage into fermentation products such as acetic acid, butyric acid and propionic acid (Russell and van soest, 1984). The high altitude of the Tibet plateau impairs rumen fermentation and increases basal metabolic rate (Qiao et al, 2013). Oxygen is a key substance for maintaining the metabolism and life of animal bodies and is necessary for normal life activities. Hypoxic environments produce a series of physiological and genomic changes that allow humans and local animals to adapt to local extreme high altitude climates (O' brien et al, 2020). Since the domestication of cattle, cattle become indispensable animal resources in plateau areas and play an important role in providing meat, milk and services. Therefore, the environmental adaptation mechanism of cattle attracts people's attention.

Yaks (Bos grunniens) and common cattle (Bos taurus) differentiate approximately 500 ten thousand years ago, but retain a high degree of homology at the genomic level (Qiu et al, 2012). Yak mainly lives in high altitude areas, especially the Xingdushushu-Himalayan area and the Qinghai-Tibet plateau area with 3000 + 5500 meters. From a physiological perspective, yaks have greater metabolic capacity than low altitude cattle (Wang et al, 2011), lower cardiopulmonary capacity, greater cardiopulmonary volume (Wiener et al, 2003). Yak and tibetan varieties have adapted to the plateau environment by expanding the number of pulmonary, bronchial arterial smooth muscles, erythrocytes, hemoglobin and hematocrit (Li et al, 2006; Ma et al, 2011). These studies lay the foundation for understanding the molecular mechanisms of human and animal adaptation to high altitude environments. Historically, ape za cows were bred 80-100 years ago by introduction of the tumor bovine (Bos indicus) ancestor from india, dan and nipol, and by cross breeding with native cattle in china (Guan et al, 2017). Tibet cattle and Japanese Ka cattle also live in plateau with altitude of more than 3500 m, and these cattle have been well adapted to high altitude anoxic environment through long-term natural selection. They also have strong foraging capacity, rough feeding resistance, high energy metabolism and strong cardiopulmonary function to ensure normal circulation and transport of blood in vivo (Dolt et al, 2007; Wang et al, 2011), which provides an ideal model for understanding the plateau hypoxia adaptive mechanism.

High-altitude areas are characterized by oxygen deficiency (the oxygen content in the air is only 50% -60% of that in plain areas), low annual average temperature (from-1 ℃ to-5 ℃), short growing season (from 6 months to 9 months) and seasonal changes in feed supply (Shao et al, 2010), resulting in lack of high-quality pasture. Therefore, under the condition of insufficient nutrient supply in plateau, how to provide metabolic function for the cattle to ensure the development of growth, development, survival and production performance is important for the regulation and adaptation of hypoxia metabolism of the plateau cattle body. In plateau regions, the energy supply is insufficient due to hypoxia, lack of feed and nutrition, and the animal body often obtains the required energy by improving glycolysis or improving the utilization rate of substrates.

The major energy source for most cells is glucose, from which Adenosine Triphosphate (ATP) is produced by glycolysis and/or oxidative metabolism. Since a deficiency in glucose causes an increase in the intracellular adenosine phosphate/adenosine triphosphate ratio, adenylate activated protein kinase (AMPK) is activated, restoring energy balance by inhibiting synthetic processes (e.g., protein or lipid synthesis) and promoting catabolic processes (e.g., glycolysis) (Xiao et al, 2017). Glycolysis is a major catabolic process in response to energy stress, the metabolic pathway by which most of the pyruvate metabolism is converted from glucose to lactate. Although normal non-proliferating cells can only undergo glycolysis under anaerobic conditions, some rely primarily on glycolysis to produce ATP and the basic unit of biosynthesis even under aerobic conditions, the so-called "aerobic glycolysis" or "wobbe effect" (Vander Heiden et al, 2009). The fructose 1.6-bisphosphate Aldolase (ALDOC) gene, aldolase, can act as a monitoring system, sensing a decrease in glucose supply before the cell energy status decreases, and activating AMPK in an AMP/ADP independent manner. Aldolase (ALDOC) is an ideal sensor for glucose utilization (Zhang et al, 2018). Under hypoxic conditions, ALDOC upregulation expression promotes glycolysis to produce ATP to meet the needs of animal energy metabolism (Leiherer et al, 2014). Under hypoxic conditions, ALDOC upregulation expression promotes glycolysis to produce ATP to meet the needs of animal energy metabolism (Leiherer et al, 2014).

Disclosure of Invention

Aiming at the prior art, the invention provides a method for screening a bovine plateau hypoxia adaptive gene ALDOC and a functional molecular marker and application thereof. The method selects the cattle varieties distributed at high altitude (the altitude is more than 1800 m) and low altitude (the altitude is less than 1500 m) from the aspects of cattle genome selection, genetic adaptation and the like, integrates three genome selection signal analysis methods of FLK, hapFLK and XPEHH by adopting a cattle 777K high-density SNP chip, compares the genome selection signals between the cattle at high altitude and in the cattle varieties, identifies candidate genes adapting to the extreme environment of high-altitude hypoxia through a combined screening strategy and bioinformatics analysis, further excavates potential functional molecular markers through selection signal verification and sequence re-testing data analysis, and establishes a corresponding detection method. The invention provides scientific basis and simple and easy detection technology for molecular breeding such as breeding of plateau hypoxia special cattle varieties; meanwhile, the method has important significance and value for the protection, evaluation and utilization of local cattle variety genetic resources.

The invention aims to provide a method for screening a bovine plateau hypoxia adaptive gene and a functional molecular marker.

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

In order to achieve the purpose, the invention relates to the following technical scheme:

in a first aspect of the present invention, a method for screening a bovine plateau hypoxia adaptive gene and a functional molecular marker is provided, the method at least comprising:

DNA samples of different altitude cattle varieties are detected and analyzed based on the SNP chip;

the method is characterized in that analysis is carried out based on FLK, hapFLK and XPEHH genome selection signal analysis methods, single nucleotide polymorphic SNPs and candidate genes subjected to significant positive selection are screened, and plateau hypoxia adaptive candidate genes and SNP molecular markers are screened by integrating gene function annotation.

Further, based on the method, the ALDOC gene is screened and identified as a key gene of the bovine plateau hypoxia adaptability; furthermore, the bovine plateau hypoxia adaptive molecular marker also comprises SNP located in the gene, including but not limited to g.20588331T > G (rs133198943A > C); g.20590099G > A (c.288C > T, aa96F > F); g, 20590664T > C; g.20591903C > A (g.20591903G > T); the single nucleotide polymorphic site g.20591903C > A has the strongest selection signal and is a functional molecular marker verified by experiments, wherein the bovine plateau hypoxia adaptive genotype is TT type or complementary sequence AA type.

In a second aspect of the present invention, there is provided the use of the above method for screening a bovine individual (population, line or breed) suitable for high altitude hypoxia survival.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme aims at the genetic characteristic of plateau hypoxia adaptation of the cattle variety, selects local cattle varieties distributed at high altitude and low altitude from the aspects of genome selection and genetic adaptation, integrates methods and strategies of various genome selection signals, bioinformatics, promoter activity analysis, gene expression analysis and the like, efficiently and accurately screens the key gene ALDOC and the functional molecular marker which adapt to the plateau hypoxia, and establishes a CRS-PCR-RFLP method of a creative enzyme cutting site by introducing mutation to detect the genotype, has reasonable design, and has the characteristics of high accuracy, simple and convenient application operation and low cost according to the detection method designed by the key gene and the functional marker;

by adopting the method provided by the technical scheme, individuals with high plateau hypoxia adaptability can be effectively screened out, and the method has important significance for breeding work and genetic improvement of cattle varieties in high-altitude regions. The technical scheme is that the molecular breeding technology is well applied to production practice once, can provide a technology for the conservation and utilization of bovine germplasm resources and the research of breed provenance, greatly saves the breeding cost and the special germplasm cultivation time, and obtains good economic and social benefits, thereby having good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 shows the consensus genes identified by FLK, hapFLK and XPEHH in the examples of the present invention (Wien diagram).

FIG. 2 shows the selection signal distribution of chromosome 19 hapFLK and XPEHH between high and low altitude bovine species in an example of the present invention.

FIG. 3 shows the SNP and haplotype evolution tree of the Chinese native cattle variety (25) differential selection region (Chr19:20.537-20.664Mb, including ALDOC gene) in the example of the present invention.

FIG. 4 is an analysis of allele frequencies of ALODC gene structure and potential functional variations thereof in different bovine populations according to an embodiment of the present invention.

FIG. 5 shows the effect of SNP (g.20591903C > A) on the promoter activity of ALDOC gene in examples of the present invention;

note: denotes P < 0.05.

FIG. 6 shows the difference in expression of liver tissue ALDOC of individual with different genotypes of SNP (g.20591903C > A) in the example of the present invention.

FIG. 7 shows the sequencing results of individuals with different genotypes of SNP (g.20591903G > T) in the examples of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

At present, few molecular biological researches on plateau hypoxia adaptation of local cattle are carried out, and no method for screening plateau hypoxia adaptation local cattle is available.

In view of the above, in one embodiment of the present invention, a method for screening a bovine plateau hypoxia adaptive gene and a functional molecular marker is provided, the method at least comprising:

DNA samples of different altitude cattle varieties are detected and analyzed based on the SNP chip;

the method is characterized in that analysis is carried out based on FLK, hapFLK and XPEHH genome selection signal analysis methods, single nucleotide polymorphic SNPs and candidate genes subjected to significant positive selection are screened, and plateau hypoxia adaptive candidate genes and SNP molecular markers are screened by integrating gene function annotation.

In another embodiment of the present invention, the method for screening the hypoxia adaptive gene and the functional molecular marker of the bovine plateau comprises:

s1, collecting and DNA extracting samples of cattle varieties at different altitudes;

s2, detecting and analyzing the SNP chip;

s3, analyzing FLK and hapFLLK genome selection signals;

s4, XPEHH genome selection signal analysis;

s5, identifying a positively selected genetic variation and a differentially selected region;

s6, screening candidate genes based on a screening strategy of the candidate genes;

s7, verifying a positive selection signal of the difference selection area;

and S8, identifying potential functional variation in the positive selection gene by using the remeasurement data.

Wherein, the steps S3 and S4 have no precedence, so the step sequence can be S3-S4 or S4-S3;

in another embodiment of the present invention, the step S1 includes:

s1.1, selecting common cattle and tumor cattle varieties at different altitudes, and mixed varieties of the common cattle and the tumor cattle;

s1.2, blood of a cow is collected, and DNA in blood tissues is extracted.

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

s2.1, analyzing the sample by using the SNP chip, and genotyping;

s2.2, filtering the SNP data, and further analyzing the remaining SNPs meeting the requirements;

s2.3 haplotype was constructed for each chromosome.

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

s3.1, respectively comparing selection signal values corresponding to SNPs of higher altitude and lower altitude groups based on an FLK and hapFLK whole genome selection signal analysis method, and acquiring difference selection signals of the SNPs between the two groups;

s3.2, respectively operating FLK and hapFLK programs by using genotype data to obtain selection signals of SNPs in the variety, and taking Nelore as a distant population; in the FLK analysis, Nelore is defined as the distant population, and K10 and nti 20;

s3.3, constructing an evolutionary tree of the whole genome and a local genome region for the selected region by using hapFLK results and Python and R scripts;

s3.4 p-value of hapFLK values were calculated by fitting a standard normal distribution of the whole genome in R.

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

s4.1, estimating XPEHH values between high-altitude and low-altitude cattle varieties, and defining the relation between physical distance and genetic distance by using 1Mb ≈ 1cM in cattle genome;

s4.2 construction of haplotypes based on ReqDel-FAST.

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

based on the results of FLK, hapFLK and XPEHH selection signal analysis, SNPs with positive selection signal values at the top 0.1% were screened separately.

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

s6.1, defining 50Kbp of the upstream and downstream SNPs obtained by identification as selected areas, and connecting overlapping areas in series to obtain a differential selection area;

s6.2, annotating a reference gene in the difference selection area or overlapped with the area by using the UCSC online website;

s6.3, screening out the FLK, hapFLK and XPEHH selection signals which are compared among high and low altitude groups, ranking 10-bit differential selection regions, respectively identifying candidate genes (such as ALDOC genes), and carrying out functional annotation.

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

s7.1, respectively constructing a whole genome and a local selection region evolutionary tree based on FLK and hapFLK analysis results, and analyzing the selection condition of the candidate gene in each cattle variety;

s7.2 the results of the comparisons between groups were further validated based on the results of the in-breed FLK and hapFLLK selection signal analysis.

In another embodiment of the present invention, the step S8 includes:

s8.1, acquiring double-end Illumina re-sequencing data, and comparing the acquired data with a reference genome (UMD3.1) respectively;

s8.2, filtering to obtain a high-quality mapping sequence, and detecting genetic variation from the high-quality mapping sequence;

s8.3, extracting genetic variation from a specific gene region, wherein the extracted regions are an upstream region and a gene region of a candidate gene;

s8.5, respectively calculating the genotype and the allele frequency of each genetic variation;

s8.6 through functional annotation, the SNP sites of potential functional variation are determined.

In another embodiment of the invention, a molecular marker for bovine plateau hypoxia adaptability, which is obtained by identification based on the method, is provided, and specifically, the ALDOC gene is a key gene of bovine plateau hypoxia adaptability and can be used as a molecular marker for bovine plateau hypoxia adaptability; meanwhile, the bovine plateau hypoxia adaptive molecular marker also comprises four mononucleotide polymorphic sites positioned on the gene, such as: g.20588331T > G (rs133198943A > C); g.20590099G > A (c.288C > T, aa96F > F); g, 20590664T > C; g.20591903C > A (g.20591903G > T). The SNP (g.20591903C > A) is a functional molecular marker verified by experiments, wherein the bovine plateau hypoxia adaptive genotype is TT type or complementary sequence AA type.

In another embodiment of the present invention, a kit for detecting the above molecular marker is provided, which can be used to screen bovine individuals (population, strain or breed) suitable for high altitude hypoxia survival; more specifically, the kit comprises a primer for detecting the single nucleotide polymorphic site; the single nucleotide polymorphic site comprises g.20588331T > G (rs133198943A > C); g.20590099G > A (c.288C > T, aa96F > F); g, 20590664T > C; any one or more of g.20591903C > A (g.20591903G > T);

in another embodiment of the invention, the invention provides a kit for detecting a single nucleotide polymorphic site g.20591903G > T, wherein the kit at least comprises the following primers:

F:5‘-GTAATTGTTTACGGTGACGC-3’(SEQ ID NO.7)；

R:5‘-GGCCTTGTTCTGATTCCTGC-3’(SEQ ID NO.8)。

in yet another embodiment of the present invention, there is provided the use of the above method for screening a bovine individual (population, line or breed) suitable for high altitude hypoxia survival.

In another embodiment of the present invention, the application method specifically comprises:

extracting blood DNA of different cattle individuals;

the kit for detecting the molecular marker is utilized to identify the genotype of the marker by a CRS-PCR-RFLP method, and identify individuals with the plateau hypoxia adaptive specific molecular marker.

In yet another embodiment of the invention, the molecular marker is a single nucleotide polymorphic site g.20591903G > T; in this case, the kit comprises at least the following primers and BstUI restriction enzyme;

F:5‘-GTAATTGTTTACGGTGACGC-3’(SEQ ID NO.7)；

R:5‘-GGCCTTGTTCTGATTCCTGC-3’(SEQ ID NO.8)。

the invention actually establishes a method for screening plateau hypoxia adaptability local cattle by using a genome direct sequencing technology. According to the invention, high-density SNP chip analysis is carried out on different varieties of local cattle, a selection signal analysis method is combined, specific SNP sites related to plateau hypoxia are found, and plateau hypoxia adaptability of the cattle can be judged by verifying single SNP sites or combination of SNP sites. The method has important significance for the molecular breeding development of cattle in plateau areas.

The present invention will be further described with reference to the following examples, but the present invention is not limited thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.

Examples

1. Acquisition and DNA extraction of high-low altitude cattle variety samples

25 domestic local cattle varieties distributed at different altitudes and 1 foreign variety are selected as distant reference groups, and 352 groups of the domestic local cattle varieties and the foreign varieties comprise common cattle, oncous cattle and mixed species of the common cattle and the oncous cattle, and blood DNA is respectively extracted. Wherein, the cattle breeds distributed in the area with the altitude lower than 1500 m form a low altitude group (LA), and the cattle breeds distributed in the area with the altitude higher than 1800 m form a high altitude group (HA). The cattle breed and grouping information are shown in table 1:

TABLE 1 cattle breed information and grouping

2. Genotyping Using Illumina high Density Bovine HD 777KSNP chips

Genotyping was performed using this chip, and the python program PEDDA _ ROW was used to extract genotype results in AB format from the genotype test result file finalreport. The chip had a total of 777,962 Single Nucleotide Polymorphisms (SNPs), and SNPs data were filtered using Plunk1.9 (http:// zzz. bwh. harvard. edu/plink 2.shtml) software to remove first 40,497 SNPs on X, Y and mitochondrial chromosomes and SNPs that did not map uniquely to UMD 3.1. Wherein SNP detection rate is lower than 90% or SNP with minimum allele frequency less than 0.05 is removed, and after filtering, the remaining 702,622 autosomal SNPs are subjected to subsequent analysis.

3. High-low altitude group and variety internal genome FLK and hapFLK selection signal analysis

Using hapflk software (https://forge-dga.jouy.inra.fr/projects/hapflk/) The FLK and hapFLK whole genome selection signal analysis method respectively compares the selection signal values corresponding to the SNPs of the higher-altitude group and the lower-altitude group, and obtains the difference selection signals of the SNPs between the two groups. In addition, the FLK and hapFLK programs were run using genotype data from 25 varieties, respectively, to obtain selection signals for SNPs in the varieties, and Nelore was used as the distant population. In the FLK analysis, Nelore is defined as the distant population, and K is 10 and ntit is 20. hapFLK results were used along with modified Python and R scripts (local _ reanalds.py and local _ trees.r) to construct evolutionary trees of whole and local genomic regions for selected regions. And the p-value of the hapFLK value was calculated by fitting a standard normal distribution of the whole genome in R.

4.XPEHH genomic selection signal analysis

XPEHH was applied in the Selscan software to estimate XPEHH values between high and low altitude varieties. The XPEHH values were normalized in each set of comparisons to have a mean and a unit squared error. We used 1Mb ≈ 1cM genetic relationships in the bovine genome. And haplotype construction was performed using ReqDel-FAST in plink1.9 and fastPHASE1.4.

5. Identification of positively selected genetic variants and differentially selected regions

The FLK, hapFLK and XPEHH selection signal analysis method is used for screening the top 0.1% of SNPs subjected to significant positive selection respectively compared with high-altitude and low-altitude cattle groups, and 701, 702 and 700 SNPs are identified respectively.

6. Differentially selected regions and identification of candidate genes

The above identified SNPs were defined as the selected regions at 50Kbp upstream and downstream, the overlapping regions were concatenated to obtain differential selection regions, and FLK, hapFLK and XPEHH identified 351, 32 and 99 differential selection regions, respectively. Finally, using online UCSC (https://genome.ucsc.edu/cgi-bin/hgTables) The identification of reference genes within or overlapping the differentially selected region resulted in the identification of 341, 50 and 142 candidate positive selection genes, respectively, that may be associated with high altitude adaptation. Among these, three methods were applied to identify 2 common (overlapping) genes; FLK and hapFLK identified 9 common genes; FLK and XPEHH identified 23 common genes; hapFLK and XPEHH identified 5 common genes in total, including ALDOC, SPAG5, KIAA0100, PIGS, SDF2 (fig. 1). Further, screening out FLK, hapFLK and XPEHH selection signals which are compared among high and low altitude groups and selecting the difference selection areas which are 10 bits before the ranking, respectively identifying candidate genes and carrying out function annotation. Among them, hapFLK values of the differential selection region (Chr19:20.537-20.664Mb) where ADLOC gene was found were ranked first; moreover, the gene was focused on finding that the gene is associated with anaerobic glycolysis. We performed subsequent analyses using the selected region and the included ALDOC gene as candidate genes.

The ADLOC gene is located on chromosome 19, and the results of genomic selection signal analysis of hapFLK and XPEHH are shown in FIG. 2. In hapFLK analysis, the differential selection region has 56 SNPs, 11 of which are located in the first 0.1% (hapFLK ≧ 17.2), and in particular SNP (rs133198943) is located in the ALODC, which has the strongest selection signal value (hapFLK ═ 20.51, P ═ 3.97E-04). In the XPEHH analysis, only 1 SNP (rs136098191) ranked the XPEHH signal value (XPEHH 3.51, P7.77E-04) at the top 0.1% (XPEHH ≧ 3.386).

7. Positive select signal verification of differentially selected regions

Respectively constructing a whole genome and a local selection region evolutionary tree by utilizing FLK (marker kinase) and hapFLK (marker kinase) analysis results in 25 varieties, analyzing the selection condition of the gene in each cattle variety, and analyzing the result as shown in figure 3, wherein in the FLK analysis in the varieties, the ALODC gene is selected most strongly in Tibet cattle (Tibet) of the plateau variety (P is 1.40E-41); the other plateau cattle variety, Aripinza broken cattle (Apeijiaza), is also strongly positively selected (P ═ 4.4E-09). In hapFLK analysis, three plateau breeds, tibetan (P7.7E-83), camu (Shigatse Humped; P4.9E-07), and apenema (P9.8E-08), were strongly selected, with the strongest signal. The results of the intravarietal selection signal analysis further confirmed the results of the comparisons between groups.

8. Identification of potential functional variations within positively selected genes using remeasured data

Double-ended Illumina re-sequencing data (data Nos.: PRJNA285834, PRJNA422979 and PRJNA396672) were downloaded from the NCBISRA database. The data contains 20 of 29 Tibet cattle and low-altitude cattle varieties (Kazakh cattle and Mongolia cattle), 10 of yaks, 14 of ruminants, 18 of wild cattle and 22 of longnius cattle. The downloaded data were aligned to the reference genome (UMD3.1) using BWA-MEM software, respectively. High quality mapping sequences were filtered using Samtools and design parameters "-view-f 4-q 20" and duplicate reads were removed with the "rmdup" parameter. Genetic variation was detected from high quality mapped sequences using GATK software. The bam file was sorted and the repeated sequence was masked using Picard (http:// broadlisting. github. io/Picard /). And extracting genetic variation from a specific gene region by using Vcftools software, wherein the extracted region is 2000Kbp upstream of the ATG of the ALDOC gene and a gene region. As a result, we identified 275 genetic variations in the population and calculated the frequency of each genotype and allele separately. By functional annotation, a total of 4 potential functional variants were identified (g.20588331T)>G，rs133198943A>C；g.20590099G>A，c.288C>T，aa96F>F；g.20590664T>C；g.20591903C>A) They have significant differences in the high and low altitude groups (fig. 4), and are potential functional variations associated with hypoxia adaptation in cattle plateau. Among them, SNP (g.20591903C)>A) In the promoter region, using AnimalTFDB (http://bioinfo.life.hust.edu.cn/AnimalTFDB/#！/) The transcription factor binding site prediction was carried out on the wild type (acggtgactcGccttcatc) and mutant sequence (acggtgactcTccttcatc), and the SNP g.20591903C was found>A(g.-1428G>T; a of translation initiation ATG +1) mutation caused a great change in transcription factor binding site, as shown in Table 2, deletion5 transcription factor (FOS, HDAC2, YBX1, YBX2, AP1) binding sites, and 1 transcription factor (FOXO3) binding site. Many of the transcription factors mentioned above are associated with hypoxia adaptation. For example, FOXO3 has been reported to be associated with hypoxia-induced endothelial apoptosis (Hu et al, Cell Signal,2018,51: 233-242). Hypoxia has been shown to inhibit hydroxylation and degradation of FOXO3 protein, resulting in accumulation and activation of FOXO3 in renal tubular epithelial cells. Hypoxia-activated HIF-1 is involved in the activation of FOXO3 and protects the kidney. In hypoxic kidneys, the stress-responsive transcription factor FOXO3 can be activated to adapt to hypoxic conditions, slowing the progression of chronic renal disease (Li et al, J Clin Invest,2019,129(6): 2374-. Hifs alters metabolic pathways by promoting anaerobic glycolysis and inhibiting oxidative phosphorylation, hypoxia activates Hifs to confer anaerobic metabolic programs on minicells, and hypoxia induces oocyte dormancy by expressing FOXO3 and Hifs (Shimamoto et al, PNAS, 2019, 116 (25): 12321-12326). FOXO 3-interfered mice soon lost fertility due to over-activation of immature oocytes (Castrillon et al, Science, 2003, 301: 215-. FOXO3 functions in reducing mitochondrial mass and oxygen consumption by inhibiting a series of nuclear-encoded mitochondrial genes (Jensen et al, EMBO J.2011, 30: 4554-. The above studies suggest that the transcription factor FOXO3 is associated with anaerobic glycolysis and hypoxia adaptation, SNP (g.20591903C)>A) It is possible to alter the expression of ALDOC by affecting the binding of transcription factors to ALDOC.

TABLE 2 ALDOC Gene SNP (g.20591903C > A or G > T) changes in wild-type and mutant sequence transcription factor binding sites

9. Effect of SNP (g.20591903C > A) on the Activity of the promoter of the ALDOC Gene

(1) Construction of recombinant plasmid: a pair of primers ALDOC-SNP20591903 is designed by using DNAs of a wild type SNP (g.20591903C > A) and a mutant type individual as templates.

5'-GGGGTACCtaccttctccatccccctct-3', SEQ ID NO.1, capital letters are restriction enzyme cutting sites and protective basic groups of restriction enzyme KpnI;

5'-CCGCTCGAGcgatgaggtggagtgactga-3', SEQ ID NO.2, capital letters are restriction sites and protective bases of restriction enzyme XhoI

Respectively amplifying fragments containing SNP (g.20591903C > A) by PCR (polymerase chain reaction), wherein the length of the amplified fragments is 327bp, constructing a wild type plasmid (pGL3-aldoc-GG) and a mutant type plasmid (pGL3-aldoc-TT) containing SNP (g.20591903C > A), and verifying the accuracy of the sequences by using product sequencing.

(2) Cell culture and transient cell transfection: the culture medium used for culturing the human liver cancer cells (HepG2) and fetal bovine fibroblasts (BFF) is a DMEM complete culture medium containing 7% FBS and 1% double antibody, the frozen cells are revived at 37 ℃, placed in a 37 ℃ and 5% incubator for culturing for 48-72 hours and then subcultured. The cultured healthy cells are subcultured to a 24-well plate and cultured for 24-48h, and cell transfection is carried out when the cell density reaches 70% -90%, wherein the method comprises the following steps:

preparing a transfection solution:

solution (50. mu.L of Opti-MEM + 1.8. mu.L of lipofectamine 2000;

solution two 50 uL Opti-MEM +800ng pGL3-basic recombinant plasmid/no-load plasmid +20ng internal reference plasmid pRL-TK and standing at room temperature for 5 min;

the solution (III) and the solution (II) are mixed in a centrifugal tube of 1.5ml and incubated for 20 min;

the original medium in the 24-well plate was discarded, washed gently twice with Opti-MEM, and 400. mu.L of Opti-MEM was added within 20min of incubation of solution I and solution II. And placing the solution (c) in a 24-well plate, culturing for 5h in a 5% incubator at 37 ℃, discarding the transfection solution, and replacing with a complete culture medium containing serum to continue culturing for 48 h. Luciferase activity was measured.

Experimental group (transfection of recombinant plasmids with different target sequences) and control group (transfection of PGL3-basic empty plasmid) were transfected separately by the above method, and BFF and HepG2 cells were transfected separately for each plasmid, and transfection was repeated 3 times.

(3) Detecting the luciferase activity: after culturing the cells for 48 hours, the 24-well plate was taken out and the medium was discarded, and the cells were washed twice with PBS. Then, 100 μ L of 1 × cell lysate PLB was added to a 24-well plate, the plate was gently shaken to accelerate cell lysis, and the cell lysis was observed in a microscope, after lysis for 15-30min, the lysed cell suspension was removed and stored in 1.5ml centrifuge tubes, respectively. And (3) putting 20 mu L of cell lysate into a 1.5ml centrifuge tube with good light transmittance, adding 100 mu L of LARII reagent, mixing gently, quickly placing in a fluorescence detector, and analyzing the activity of the firefly luciferase. And taking out the centrifuge tube from the fluorescence detector, quickly adding 100 mu L of Stop & GloTM reagent, quickly quenching the activity of firefly luciferase, activating the renilla luciferase reaction of the internal reference plasmid pRL-TK, and placing the renilla luciferase reaction product in the fluorescence detector for analyzing the activity of the renilla luciferase. The ratio of firefly Luciferase Activity to Renilla Luciferase Activity is the Relative Luciferase Activity (RLA), with the RLA results being the average of three independent replicates. The results are shown in FIG. 5, demonstrating that TT mutations enhance ALDOC promoter activity.

10. Effect of SNP (g.20591903C > A) on hepatic tissue ALDOC expression

(1) Liver tissues of 3 cattle with GG and TT genotypes are selected respectively, and RNA is extracted.

(2) A pair of primers ALDOC-mRNA is designed, and the size of the product is 249 bp.

F:5’-agtacgttacagagaaggtcct-3’SEQ ID NO.3

R:5’-cattgaggttgagagatgcct-3’SEQ ID NO.4

And analyzing the expression condition of the ALDOC gene in the liver tissue by using fluorescence quantitative QPCR. The results are shown in FIG. 6 below.

11. SNP sequencing identification method of plateau hypoxia adaptive gene ALDOC

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

(2) A pair of PCR amplification primers containing the SNP (g.20591903G > T) site was designed.

F:5’-taccttctccatccccctct-3’(SEQ ID NO.5)

R:5’-cgatgaggtggagtgactga-3’(SEQ ID NO.6)

(3) PCR amplification was performed in a 25. mu.L PCR reaction system including 0.5. mu.L (10. mu. mol/L) of the forward primer, 0.5. mu.L (10. mu. mol/L) of the reverse primer, 1. mu.L (50. mu. mol/L) of the DNA template, ddH₂O10.5. mu.L, 2 XTaq PCR Master Mix 12.5. mu.L, reaction conditions were: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, wherein the steps are performed for 35 cycles, and finally extension at 72 ℃ for 10min, and the target fragment length is 327 bp. The PCR product was detected by electrophoresis on a 1% agarose gel.

(4) And (3) directly sequencing the amplification product, and analyzing and comparing according to a cattle ALDOC gene sequence published by NCBI, wherein individuals with G > T mutation, namely individuals with the genotype of TT belong to plateau hypoxia-adaptive cattle (see figure 7).

12. SNP (g.20591903G > T) gene detection method of plateau hypoxia adaptive gene ALDOC

(1) Different cattle varieties are selected, and blood DNA is extracted.

(2) Primers are designed aiming at specific SNP (g.20591903G > T) sites, and restriction enzyme cutting sites BstUI are generated by introducing 1 mutation into an upstream primer, so that BstUI restriction enzymes can cut PCR products with different mutations into fragments with different lengths.

F:5’-GTAATTGTTTACGGTGACGC-3’(SEQ ID NO.7)

R:5’-GGCCTTGTTCTGATTCCTGC-3’(SEQ ID NO.8)

And (3) PCR amplification: PCR amplification was carried out in a 25. mu.L PCR reaction system including 0.5. mu.L (10. mu. mol/L) of the forward primer, 0.5. mu.L (10. mu. mol/L) of the reverse primer, 1. mu.L (50. mu. mol/L) of the DNA template, ddH2O 10.5.5. mu.L, 2 XTaq PCR Master Mix 12.5. mu.L under the following conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, wherein the steps are performed for 35 cycles, and finally extension at 72 ℃ for 10min, and the target fragment is 104bp in length.

CRS-PCR-RFLP genotyping: carrying out enzyme digestion on the PCR product by using restriction enzyme BstUI, detecting by 2.5% agarose gel electrophoresis after enzyme digestion, and separating two bands of 84bp and 20bp from a wild homozygous individual, wherein only one band, namely 84bp, is displayed in gel because the 20bp fragment is smaller); the heterozygote individual can separate four bands of 104bp, 84bp and 20bp, wherein, the 20bp segment is smaller, so two bands of 104bp and 84bp are displayed in the gel; a band of 104bp can be separated from individuals with homozygous mutant.

Secondly, the embodiment also discloses a kit containing the primer and the enzyme.

The kit also comprises a PCR amplification reaction reagent and an enzyme digestion reaction reagent.

Specifically, the PCR amplification reaction reagent comprises dNTP (25mM each), MgCl₂(25mM)、PCR Bμffer、ddH₂O, etc.;

the enzyme digestion reaction comprises ddH₂O, BstUI enzyme Buffer, BstUI enzyme (10U/. mu.l).

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shandong OersX animal husbandry Co., Ltd, Dairy research center of Shandong province academy of agricultural sciences

<120> method for screening bovine plateau hypoxia adaptive gene ALDOC and functional molecular marker and application thereof

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<170> PatentIn version 3.3

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