Application of a single nucleotide genetic marker in the breeding of high-altitude adapted yellow cattle

Abstract

This invention discloses the application of single nucleotide genetic markers in the breeding of Tibetan cattle adapted to high altitudes. Using whole-genome infiltration analysis, the whole-genome SNPs of Tibetan cattle were analyzed, revealing that three single nucleotide variations on the promoter of the Tibetan cattle EGLN1 gene may be associated with high-altitude adaptation. Furthermore, molecular markers for breeding high-altitude adapted Tibetan cattle were identified, which can be used to rapidly establish high-altitude adapted cattle populations, providing a basis for breeding cattle breeds adapted to high-altitude environments.

Description

Technical Field

[0001] This invention relates to cattle ( Bos taurus Genomic variations related to high-altitude adaptation specifically involve the discovery of SNPs associated with high-altitude adaptation in cattle at the DNA level and their application in the breeding of yellow cattle. Background Technology

[0002] The Tibetan cattle (also known as Tibetan cattle) in the Qinghai-Tibet Plateau region include breeds such as Apei Gyaltsen, Shigatse, Zhangmu, Diqing, Yushu, Dingjie, and Ganzi (or local herds). Tibetan cattle are small breeds, descendants of the aurochs. Due to the complex geographical barriers of the Qinghai-Tibet Plateau, they have never migrated outside of it, making them one of the oldest cattle breeds in the world. Furthermore, because they have lived in the high-altitude region for a long time, they have gradually adapted morphologically and physiologically to the extreme environment of high altitude and low oxygen.

[0003] Single nucleotide polymorphism (SNP) is a phenomenon where a single nucleotide differs in the allelic sequence of the genomic DNA of different individuals of the same species. SNPs are the most widespread type of variation in an organism's genome, caused by the insertion, deletion, transition, or transversion of a single nucleotide. DNA of the same length can form different conformations due to differences in sequence or even a single base, and these differences can affect DNA expression and function.

[0004] EGLN1(Egl-9 Family Hypoxia-Inducible Factor 1) is a gene encoding a protein that plays a crucial role in high-altitude adaptation. EGLN1 is an oxygen-sensing protein that monitors changes in intracellular oxygen concentration, helping to regulate cellular oxygen perception and thus enabling the body to better adapt to the hypoxic environment of high altitudes. Under hypoxic conditions, EGLN1 regulates intracellular oxygen signaling pathways by hydroxylating oxygen-sensitive leucine residues. For example, EGLN1 participates in regulating the stability of the HIF-1α (Hypoxia-Inducible Factor 1α) protein. Under normal oxygen levels, EGLN1 degrades HIF-1α through hydroxylation, but in hypoxic environments, EGLN1 activity decreases, leading to increased stability of the HIF-1α protein. This activates a series of genes related to hypoxia adaptation, such as angiogenesis and erythrocyte proliferation. Therefore, EGLN1 expression is often low in high-altitude species. EGLN1 is also involved in the regulation of heme synthesis. In hypoxic environments, it affects heme synthesis by modulating the expression of the HO-1 (Heme Oxygenase-1) gene in the liver. The function of EGLN1 becomes particularly important in high-altitude environments, and varies among different animals. EGLN1 Genetic variations may be related to adaptation to high-altitude regions.

[0005] Although the abundant cattle breeds (or populations) in the Qinghai-Tibet Plateau region represent a valuable genetic resource for breeding new beef cattle breeds with strong cold resistance and other adverse conditions, there are currently no reports of discovering genetic variations related to high-altitude adaptation through whole-genome resequencing analysis of Tibetan cattle at the DNA level, combined with collected whole-genome data from yaks and low-altitude distributed cattle. This may be because current research has not been able to link candidate gene variations to traits related to high-altitude adaptation developed through animal evolution; it has only examined the distribution characteristics of variations in different adapted populations and their impact on gene function.

[0006] In addition, some studies have pointed out that people EGLN1 The four SNP loci linked above, rs2790882, rs480902, rs2486736 and rs2739513, are molecular markers that significantly affect high altitude (distribution of populations at different high altitudes), lung function and blood oxygen saturation. However, humans and ruminants have significant differences in morphology, physiology and ways of adapting to the environment, and may have different adaptive selection loci. Summary of the Invention

[0007] The purpose of this invention is to provide an application of single nucleotide genetic markers in the breeding of high-altitude adapted yellow cattle. By utilizing the discovered Tibetan cattle-specific SNPs, the potential of individuals to adapt to high altitudes can be determined, and a high-altitude adapted cattle population can be quickly established, thereby contributing to the breeding of high-altitude adapted yellow cattle breeds.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Firstly, this study provides a candidate genomic region related to high-altitude adaptation in cattle. Based on gene data obtained through whole-genome sequencing and the U10 method used to detect genomic infiltration of wild yaks into Tibetan cattle (specifically a herd of 75 Tibetan cattle) from the Qinghai-Tibet Plateau, SNPs in the infiltrated region were extracted. Subsequently, the SNP frequencies of Tibetan cattle and low-altitude distributed Tibetan cattle were statistically analyzed to determine the SNPs in Tibetan cattle. EGLN1 Three highly linked SNP sites in the gene promoter region, namely 28:4151028, 28:4150843 and 28:4150859 (the bovine reference genome is ARS-UCD1.2), may contain single nucleotide variants associated with bovine high-altitude adaptation, thus identifying the infiltrated region as a candidate genomic region associated with bovine high-altitude adaptation.

[0010] Preferred subjects were six cattle populations in the Qinghai-Tibet Plateau region (Changdu cattle, Diqing cattle, Dingjie cattle, Yushu cattle, Shigatse cattle, and Zhangmu cattle) as high-altitude distributed cattle (specifically Tibetan cattle). Wild yaks were used as the introgression source population, and water buffalo as the out-group. The U10 method was used to identify an introgression region on chromosome 28 of the Tibetan cattle genome derived from wild yaks. Annotation of this introgression region (using the ARS-UCD1.2 annotation file) revealed that this region contains hypoxia adaptation genes. EGLN1 and its startup subregion.

[0011] Preferably, combining frequency distribution and linkage disequilibrium analysis, the SNP extraction results of the infiltration region are used to determine the location of the SNPs in the infiltration region. EGLN1 Three mutation sites in the promoter region, namely 28:4151028, 28:4150843, and 28:4150859, were selected as candidate sites. The mutation types of the three candidate sites were identified as follows: the SNP site at position 4151028 on chromosome 28 was a G>C mutation, the SNP site at position 4150843 was a C>G mutation, and the SNP site at position 4150859 was a T>C mutation.

[0012] Preferably, based on the strong linkage of the three variant sites 28:4151028, 28:4150843 and 28:4150859, dual-luciferase vectors were constructed for the yak type (genotypes CC, GG, CC consistent with wild yaks) and the cattle type (genotypes GG, CC, TT consistent with cattle) for the corresponding three SNP sites, and expression experiments were conducted using different cells. The results showed that the dual-luciferase activity of the yak type was significantly reduced.

[0013] Preferably, by testing and analyzing the association between high-altitude and low-altitude distributed yellow cattle populations and the above SNPs, and by selecting Tibetan cattle populations that are geographically close (but have different distribution altitudes) (such as Dingjie cattle and Shigatse cattle) for corresponding association analysis, it was determined that the single nucleotide variant unique to high-altitude distributed yellow cattle (specifically Tibetan cattle), represented by the G>C mutation at position 4151028 on chromosome 28 of the bovine reference genome, is related to the adaptation of cattle to high altitude.

[0014] Secondly, a method for detecting candidate genomic region SNPs related to bovine high-altitude adaptation is provided, comprising the following steps:

[0015] Using bovine genomic DNA as a template, PCR amplification was performed. EGLN1 Gene promoter region fragment (or amplification) EGLN1 (start sub-region segment), and then on the located in the EGLN1 The genotypes of SNP sites in the gene promoter region are identified (e.g., by DNA sequencing, i.e., sequencing and comparing the amplified products; other methods may also be used). The SNP sites are one or more of the SNP sites located at positions 4151028, 4150843, and 4150859 on chromosome 28 of the bovine reference genome. The bovine reference genome is ARS-UCD1.2. The SNP site at position 4151028 is a G>C mutation, the SNP site at position 4150843 is a C>G mutation, and the SNP site at position 4150859 is a T>C mutation (i.e., the SNP sites are one or more of 28:4151028 G>C, 28:4150843 C>G, and 28:4150859 T>C).

[0016] Preferably, the cattle to be tested are from Tibetan cattle groups such as Dingjie cattle.

[0017] Preferably, the template for designing the PCR amplification primers can refer to the following region: 28:4150028-28:4151859.

[0018] Thirdly, a detection kit for candidate genomic region SNPs related to bovine high-altitude adaptation is provided, including a kit for specifically amplifying the SNPs of the target bovine region. EGLN1Primers (e.g., KASP primers) for identifying SNP sites in the gene promoter region and the corresponding SNP genotypes, wherein the SNP sites are one or more of the SNP sites located at positions 4151028, 4150843, and 4150859 on chromosome 28 of the bovine reference genome; the bovine reference genome is ARS-UCD1.2, and the SNP site at position 4151028 is a G>C mutation, the SNP site at position 4150843 is a C>G mutation, and the SNP site at position 4150859 is a T>C mutation (i.e., the SNP sites are one or more of 28:4151028 G>C, 28:4150843 C>G, and 28:4150859 T>C).

[0019] Fourthly, an application of a SNP locus related to high-altitude adaptation in cattle in marker-assisted selection breeding is provided, wherein the SNP locus is located at position 4151028 on chromosome 28 of the bovine reference genome; the bovine reference genome is ARS-UCD1.2, and the SNP locus at position 4151028 is a G>C mutation.

[0020] This invention also provides the application of the above-mentioned detection method in marker-assisted selection breeding of cattle.

[0021] Preferably, the high-altitude adaptability of cattle specifically refers to the ability of cattle to survive and maintain stable production (milk and meat production) performance in environments above 3000 meters altitude.

[0022] Preferably, the CG genotype at the SNP locus at position 4151028 is a molecular marker that can improve the high-altitude adaptability of cattle.

[0023] Fifthly, a method for breeding high-altitude adapted yellow cattle using single nucleotide genetic markers is provided, comprising the following steps:

[0024] Using bovine genomic DNA as a template, PCR amplification was performed. EGLN1 Gene promoter region fragment (or amplification) EGLN1 (a partial fragment of the gene promoter region), and then located in the... EGLN1 Genotypes of SNP sites in the gene promoter region were identified, and then a high-altitude adapted yellow cattle population was established based on the corresponding genotypes of the cattle to be tested. The SNP site is the SNP site at position 4151028 on chromosome 28 of the bovine reference genome; the bovine reference genome is ARS-UCD1.2, and the SNP site at position 4151028 is a G>C mutation (i.e., the SNP site is 28:4151028 G>C, and the corresponding genotype is CG genotype).

[0025] Preferably, the cattle to be tested are from Tibetan cattle groups such as Dingjie cattle.

[0026] The beneficial effects of this invention are reflected in:

[0027] This invention uses methods such as genome resequencing and genome infiltration detection to identify and discover traits associated with high-altitude adaptation in Tibetan cattle populations. EGLN1 The gene promoter region variant site 28:4151028 G>C and highly linked variant sites (e.g., 28:4150843 C>G and 28:4150859 T>C) can be identified. By identifying the genotype of these sites, the carrier status of mutant alleles at sites related to high-altitude adaptation can be determined. Molecular markers at the corresponding sites can be used to quickly screen individuals for constructing a high-altitude adapted population, which is of great significance for the breeding of high-yielding beef and dairy cattle breeds in plateau areas. Attached Figure Description

[0028] Figure 1 This represents the penetration region obtained using the U10 method.

[0029] Figure 2 for EGLN1 Linkage analysis heatmap of high-frequency gene variant sites in Tibetan cattle population (including the reference genome region in the range of 28:4150446-28:4153795).

[0030] Figure 3 for EGLN1 Results of dual-luciferase assay for expression levels of yak and cattle genotypes. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0032] (I) Exploring candidate variants related to high-altitude adaptation in Tibetan cattle using whole-genome resequencing

[0033] (1) Collect whole genome sequencing data of 75 cattle from six cattle groups on the Qinghai-Tibet Plateau (7 from Changdu, 9 from Diqing, 30 from Dingjie, 22 from Yushu, 5 from Shigatse, and 2 from Zhangmu, which together constitute the experimental Tibetan cattle group) (see Table 1). Whole-genome sequencing data for Changdu and Zhangmu cattle were collected from publicly available databases. Samples of Diqing cattle were collected in June 2018 from Diqing Tibetan Autonomous Prefecture, Yunnan Province; samples of Dingjie and Shigatse cattle were collected in June 2018 from Shigatse City, Tibet Autonomous Region; and samples of Yushu cattle were collected in June 2018 from Yushu Tibetan Autonomous Prefecture, Qinghai Province. All collected samples were brought back to the laboratory for DNA extraction and then resequencing. At the same time, whole-genome sequencing data for 4 wild yaks, 2 water buffaloes, and 107 low-altitude distributed yellow cattle (Angus, Anxi, Qaidam, Han, Hereford, Holstein, Jersey, Kazakh, Mongolian, Simmental, Yakut, and Yanbian cattle) were collected from publicly available databases.

[0034] Table 1. Data Collection for Analysis

[0035]

[0036] (2) Genome alignment: The resequencing results of each cow were aligned to the bovine reference genome ARS-UCD1.2 (NCBI version number GCF_002263795.1) using BWA software.

[0037] (3) Use software such as Picard and GATK to detect all SNP sites in all cattle and annotate them.

[0038] (4) Using wild yak as the infiltrated source population and buffalo as the out-population, the U10 method was used to detect the infiltrated regions across the entire genome. The infiltrated regions located at 28:3450001-28:4670000 in the bovine reference genome were obtained. Combined with the functional query of candidate genes, the infiltrated regions were located on chromosome 28. EGLN1 Isogenes ( Figure 1 ).

[0039] (5) Using GATK software, SNPs were extracted from the infiltration area, and the SNP frequencies of Tibetan cattle and low-altitude yellow cattle were statistically analyzed. EGLN1 The promoter region contains three high-frequency mutation sites in Diqing and Dingjie cattle: 28:4151028, 28:4150843, and 28:4150859 (on chromosome 28, position 4151028 is a G>C mutation SNP, position 4150843 is a C>G mutation SNP, and position 4150859 is a T>C mutation SNP), and these three SNPs are highly linked. Figure 2 ).

[0040] (6) The yak type after mutation at the above three SNP sites ( EGLN1 -Yak: CC, GG, CC) and unmutated yellow cattle type ( EGLN1 -Taurine: GG, CC, TT), verified by dual-luciferase assays in RAW264.7 (mouse mononuclear macrophage cell line) and HeLa (human cervical cancer cell line), showed that variations in these three SNPs significantly reduced dual-luciferase activity. Figure 3 That is, the mutations in these promoter regions will be significantly reduced. EGLN1 Gene expression.

[0041] (7) Genotyping was performed on the major causal variation sites (specifically, the three SNP sites: 28:4151028 G>C, 28:4150843 C>G, and 28:4150859 T>C) (Table 3), and frequency statistics were performed on cattle distributed at different altitudes (the high-altitude cattle samples were from the aforementioned Tibetan cattle population) (Table 2). The results showed that... EGLN1 The variations at these three sites in the promoter region are only present in Tibetan cattle populations, and their frequencies are relatively stable. Combined with previous infiltration analysis and dual-luciferase activity analysis, this suggests that these three variations originate from Tibetan cattle-specific variations infiltrated by wild yaks, and that the mutations can reduce... EGLN1 Gene expression.

[0042] Table 2. EGLN1 Promoter subregion SNPs and their individual frequency statistics

[0043]

[0044] Table 3. EGLN1 Genotype and allele frequency distribution of three SNP sites in the promoter region in different cattle populations

[0045]

[0046] The above results indicate that hypoxia adaptation genes exist in Tibetan cattle populations. EGLN1 The three strongly linked Tibetan cattle-specific SNP sites (28:4151028 G>C, 28:4150843 C>G, and 28:4150859 T>C) found on the promoter were derived from introgression by wild yaks. The frequencies of these variants in the Tibetan cattle population were stable at 0.25, 0.26, and 0.26, respectively. However, dual-luciferase assays showed that the yak type significantly reduced these mutations compared to the yellow cattle type. EGLN1 Gene expression also suggests low expression EGLN1 This may be related to the fact that Tibetan cattle are better adapted to the plateau environment (high altitude).

[0047] (ii) Based on EGLN1 Application of promoter region variation breeding in high-altitude adapted yellow cattle

[0048] (1) EGLN1 The genotype frequencies of the three SNP sites in the promoter region and their correlation with sampling altitude were determined by a one-way ANOVA test. The results are shown in Table 4.

[0049] Table 4. Association analysis between altitude distribution of Tibetan cattle and SNP loci

[0050]

[0051] (2) EGLN1 The results of the one-way ANOVA test of the genotypes of the three SNP loci in the promoter region and the population altitude are shown in Tables 5-1, 5-2 and 5-3.

[0052] Table 5-1. Association analysis between altitude (unit: m) of cattle population distribution and SNP loci (Part 1)

[0053]

[0054] Note: Different letters on the average altitude indicate significant differences, while the same letter indicates no significant difference.

[0055] Table 5-2. Association analysis between the altitude (unit: m) of cattle population distribution and SNP loci (Part II)

[0056]

[0057] Note: Different letters on the average altitude indicate significant differences, while the same letter indicates no significant difference.

[0058] Table 5-3. Association analysis between altitude (unit: m) of cattle population distribution and SNP loci (Part 3)

[0059]

[0060] Note: No individuals with the homozygous mutant genotype were detected at any of the different SNP loci; the distribution altitude of Dingjie cattle is approximately 4610 meters, and the distribution altitude of Shigatse cattle is approximately 3900 meters.

[0061] The above results indicate that in hypoxia adaptation genes EGLN1 Among the three strongly linked Tibetan cattle-specific SNP sites found on the promoter, the mutant heterozygous type of the SNP site 28:4151028 G>C (i.e., the CG genotype) can serve as an important candidate molecular marker (SNP marker) for marker-assisted selection to improve the adaptability of cattle to high altitudes in the breeding of yellow cattle. For example, it can be used for the early and rapid establishment of dominant cattle populations.

[0062] (3) Rapid detection of molecular markers in cattle herds and selection of individuals adapted to high altitudes

[0063] Using inclusion EGLN1 The reference genome sequence of the gene promoter was used as a template to design PCR amplification primers. The candidate SNP marker location (i.e., 28:4151028 G>C) was selected, and extensions were made to both sides by a certain length (e.g., 500 bp). Genomic DNA from blood collected from individual cattle was used as a template for PCR amplification, and the amplified products were sequenced. The sequencing results were then compared with the reference genome sequence to identify the genotype of the individual cattle at that SNP site. If the genotype was CG, it indicated that the corresponding individual cattle carried the candidate molecular marker. Individuals with this genotype could be collected as genetic material for breeding yellow cattle, allowing the selected population to adapt to the extreme high-altitude environment.

Claims

1. The application of a substance for detecting single nucleotide polymorphisms associated with high-altitude adaptation in cattle in the assisted selection of cattle with high-altitude adaptability, characterized in that: The single nucleotide polymorphism site is located at position 4151028 on chromosome 28 of the bovine reference genome ARS-UCD1.2, and the nucleotide at this site is a G>C mutation; To improve the high-altitude adaptability of cattle, individuals with the CG genotype at the single nucleotide polymorphism site were selected; The cattle in question are those from the Qinghai-Tibet Plateau region.

2. A method for assisting in the breeding of high-altitude adaptable yellow cattle, characterized in that: Includes the following steps: Using the genomic DNA of the cattle to be tested as a template, PCR amplification was performed. EGLN1 Gene promoter region or a portion thereof, and then located in the EGLN1 The genotype of the single nucleotide polymorphism (SNP) site in the gene promoter region was identified. The SNP site is located at position 4151028 on chromosome 28 of the bovine reference genome ARS-UCD1.2, and the nucleotide at this site is a G>C mutation. To improve the high-altitude adaptability of cattle, individuals with the CG genotype at the single nucleotide polymorphism site were selected; The cattle in question are those from the Qinghai-Tibet Plateau region.

Images