Detection method and application of molecular marker related to cattle cold adaptability

Abstract

The invention discloses a detection method and application of a molecular marker related to cattle cold adaptability. On the basis of large-batch whole genome re-sequencing data of northern cattle and southern cattle, a genome selection signal analysis method is adopted, and multi-omics verification such as transcriptome and epime is combined, so that the molecular marker related to the cold environment adaptability is screened out. The molecular marker can be used for cold adaptive molecular breeding of cattle population, and provides scientific basis and technical support for genetic improvement and resource utilization of cattle species adaptive to cold environment.

Description

Technical Field

[0001] This invention belongs to the field of animal genetics and breeding and biotechnology, and involves screening molecular markers related to cold adaptation in cattle. In particular, it involves identifying and applying single nucleotide polymorphism (SNP) genetic markers based on whole-genome selection signal analysis and multi-omics validation to assist in the breeding of cattle populations with cold environment adaptability. Background Technology

[0002] Single nucleotide polymorphism (SNP) is the phenomenon where a single nucleotide differs in the allele sequence of the genomic DNA of different individuals of the same species. SNP is the most widespread type of variation in an organism's genome, caused by the insertion, deletion, transition, and transversion of a single nucleotide.

[0003] With the intensification of global climate change, extreme environments pose severe challenges to livestock production and threaten food security based on livestock farming. Therefore, animal adaptability to the environment is particularly important. Although the phenotypes of certain animal breeds or populations that have undergone long-term adaptation can be used as breeding materials for research (see CN117625806A), the adaptability of animals to specific environments is often not systematically assessed. This not only hinders the discovery of molecular markers related to desirable traits (such as heat tolerance and cold tolerance) but also limits their widespread application in breeding.

[0004] Taking cold adaptation as an example, current research on the ability of domestic cattle to adapt to cold environments still has significant shortcomings, including: existing studies mainly rely on preliminary analysis based on SNP chips, which involves a large number of detection sites, complex operation, and a lack of validation of the association between molecular markers and environmental adaptation phenotypes (see CN116590422A). In addition, the sample size of studies is limited, a high-quality genomic data system covering cattle breeds with different geographical distributions has not yet been constructed, and comprehensive validation by combining multi-omics data has not been carried out. This, to some extent, limits the systematic analysis of genetic variations related to cold adaptation in cattle.

[0005] In existing research, LPGAT1 Genes are believed to be related to lipid metabolism and energy metabolism processes. In cold environments, they may participate in the body's response to low-temperature stimuli by regulating physiological processes such as lipid metabolism pathways and cell membrane fluidity (see "Wang Huijuan. Differential gene expression in brown adipose tissue of Burmese tree shrew under cold acclimatization based on transcriptome sequencing. Yunnan Normal University, 2021."). However, LPGAT1 The gene has not yet been identified as a candidate cold-resistant gene or cold-resistant gene in the relevant research fields of animal breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for detecting molecular markers related to bovine cold adaptation, which can locate candidate genomic regions (especially...) through detection. LPGAT1 Single nucleotide polymorphism (SNP) sites on genes can be used to assist in the selection of cold-adapted cattle at the DNA level, thereby rapidly screening and establishing cattle populations with cold-resistant potential (such as the core population of yellow cattle).

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Firstly, a method for screening candidate genomic regions related to bovine cold adaptation is provided, the method comprising the following steps: Based on whole-genome resequencing data, the DCMS method was used to detect selected contrast signals in the genomes of Northern and Southern Yellow Cattle, and selected regions with high overall selection signal intensity (e.g., the top 0.5%) were extracted. Candidate genomic regions (e.g., 71.56 MB to 71.71 MB on chromosome 16 of the bovine reference genome) were determined based on multiple identified significant selected regions. The bovine reference genome was ARS-UCD1.2 (NCBI version number GCF_002263795.1).

[0008] Preferably, gene function annotation analysis is performed on the gene regions of the candidate group, and candidate genes are located.

[0009] Preferably, the candidate gene is LPGAT1 Gene.

[0010] Preferably, the screening method further includes the following steps: by comparing the SNP allele frequencies of southern and northern cattle in the selected region, identifying key SNP sites that may exist in northern cattle related to cold adaptation.

[0011] Preferably, the key SNP site is selected from those located on chromosome 16. LPGAT1 The dominant genotype in Northern Yellow Cattle is GG at the three SNP loci 16:71634198, 16:71593153, and 16:71699738 in the gene intron region.

[0012] Preferably, the screening method further includes the following step: performing a systematic statistical test on the association between the environmental adaptability phenotype and the above three SNP loci in the southern and northern yellow cattle populations.

[0013] Secondly, a method for detecting molecular markers related to bovine cold adaptation is provided, comprising the following steps: Using the bovine genomic DNA to be tested as a template, partial fragments of candidate genomic regions (specifically referring to 71.56MB to 71.71MB on chromosome 16 of the bovine reference genome) were amplified by PCR (e.g., LPGAT1 The amplified fragments are a portion of the first intron region of the gene, and the genotypes of the SNP sites contained in the amplified fragments are identified (e.g., by electrophoresis, sequencing, and alignment of the amplified products). The bovine reference genome is ARS-UCD1.2 (NCBI version number GCF_002263795.1).

[0014] Preferably, the SNP site is located at position 71634198 on chromosome 16 of the bovine reference genome, and the reference allele of this SNP site (i.e., 16:71634198) is G, and the mutant allele is C.

[0015] Preferably, the amplification primers used in the PCR can be designed with reference to genomic region 16:71633698–71634698.

[0016] Preferably, the cattle to be tested are derived from yellow cattle.

[0017] Thirdly, a detection kit for molecular markers related to bovine cold adaptation is provided. This kit includes primers for amplifying SNP sites located in candidate genomic regions (specifically, 71.56 MB to 71.71 MB on chromosome 16 of the bovine reference genome). The bovine reference genome is ARS-UCD1.2 (NCBI version number GCF_002263795.1).

[0018] Preferably, the SNP site includes position 71634198 on chromosome 16 of the bovine reference genome.

[0019] Fourthly, this invention provides an application of SNP loci associated with bovine cold adaptation in marker-assisted selection breeding of cattle, wherein the SNP loci are located within a candidate genomic region (specifically, 71.56 MB to 71.71 MB on chromosome 16 of the bovine reference genome) (e.g., located in...). LPGAT1 (The first intron region of the gene). The bovine reference genome is ARS-UCD1.2 (NCBI version number GCF_002263795.1).

[0020] Preferably, the SNP site is located at position 71634198 on chromosome 16 of the bovine reference genome, with the reference allele being G and the mutant allele being C.

[0021] Preferably, the genotype GG at the SNP locus is significantly associated with bovine cold adaptation.

[0022] Preferred individuals of yellow cattle with the GG genotype are superior to those with the GC or CC genotypes in terms of cold resistance or cold resistance potential.

[0023] This invention also provides the application of the above-mentioned method for detecting molecular markers related to bovine cold adaptability in bovine marker-assisted selection breeding.

[0024] Preferably, all individual cattle to be tested are screened based on the GG genotype and a core population of yellow cattle with cold resistance potential is established, thereby accelerating the genetic improvement and molecular breeding process of yellow cattle breeds adapted to cold environments.

[0025] Fifthly, a method for breeding cold-adapted yellow cattle using single nucleotide genetic markers is provided, comprising the following steps: Using bovine genomic DNA as a template, partial fragments of candidate genomic regions (specifically, 71.56 MB to 71.71 MB on chromosome 16 of the bovine reference genome) were amplified by PCR. Genotypes of SNP loci contained in the amplified fragments were identified (e.g., by electrophoresis, sequencing, and alignment of the amplified products). Individuals with genotypes associated with cold adaptation (e.g., GG) at the corresponding loci were screened from all tested cattle. The bovine reference genome was ARS-UCD1.2 (NCBI version number GCF_002263795.1).

[0026] Preferably, the SNP site is located at position 71634198 on chromosome 16 of the bovine reference genome.

[0027] The beneficial effects of this invention are reflected in: This invention, based on extensive bovine whole-genome resequencing data, first screens candidate genomic regions associated with cold adaptation in Northern Yellow Cattle populations using selection signals. Then, it identifies key SNP loci using population-based allele frequency analysis. Finally, through association analysis and multi-omics functional validation, it identifies functional variant sites in the candidate genomic regions (e.g., on chromosome 16). LPGAT1 The intron regions of the gene are significantly associated with cold adaptability (71634198). This invention can rapidly establish a cold-resistant cattle population by detecting the genotype of candidate loci (specifically GG), thereby accelerating the genetic improvement and molecular breeding process of cattle breeds adapted to cold environments, and has important application value and promotion prospects. Attached Figure Description

[0028] Figure 1 The selected region obtained by the DCMS method.

[0029] Figure 2 for LPGAT1An integrated view of the regulatory functions of dominant alleles at the multi-omics level; ATAC in the figure represents chromatin accessibility sequencing data, and also integrates histone modification information, including H3K27ac and H3K27me3.

[0030] Figure 3 The sequence alignment of the known binding site (motif) of transcription factor KLF15 with the candidate allele region is shown in the figure; the potential influence of the reference allele G at site 71634198 on chromosome 16 on the binding ability of KLF15 is shown in the figure.

[0031] Figure 4 for LPGAT1 Results of gene expression levels in multi-tissue transcriptomes of cattle in the north and south; in the figure, Tauine represents northern yellow cattle and Indicine represents southern yellow cattle. Detailed Implementation

[0032] 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.

[0033] (i) Exploring candidate variants associated with bovine cold adaptation using whole-genome resequencing (1) Obtain whole genome sequencing data In this experiment, ear tissues from 14 domestic cattle breeds (specifically, 7 breeds of Northern Yellow Cattle and 7 breeds of Southern Yellow Cattle, see Tables 1-1 and 1-2) were first collected and resequencing was performed.

[0034] Table 1-1. Sample Collection for Analytical Data

[0035] Table 1-2. Sample collection for analysis data

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

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

[0038] (4) Using northern yellow cattle as the target population and southern yellow cattle as the control population, the DCMS method was used to detect selection signals in the whole genome, and the selected regions with the highest overall selection signal intensity were extracted. The results showed that there were several significant selected regions on chromosome 16 of the reference genome (71.56MB to 71.71MB). Gene function annotation analysis of this region (Table 2) was performed, and candidate genes related to cold adaptation were finally located. LPGAT1 ( Figure 1 ).

[0039] Table 2. (and) LPGAT1 Statistics of gene-related candidate regions

[0040] (5) The SNPs in the selected regions identified by the DCMS method were extracted using GATK software, and the allele frequencies at each site were statistically analyzed. Three potential candidate sites were identified, located at position 71634198 on chromosome 16 (specifically located at...). LPGAT1 (first intron of the gene), position 71593153 (specifically located at) LPGAT1 (first intron of the gene) and position 71,699,738 (specifically located at) LPGAT1 (Seventh intron of the gene). Statistical analysis was performed on the allele frequencies of these three loci in northern and southern cattle populations, and the results are shown in Tables 3-1, 3-2, and 3-3.

[0041] Table 3-1. LPGAT1 Statistics on SNPs in intron regions of genes and their individual frequencies

[0042] Table 3-2. LPGAT1 Statistics on SNPs in intron regions of genes and their individual frequencies

[0043] Table 3-3. LPGAT1 Statistics on SNPs in intron regions of genes and their individual frequencies

[0044] The results indicate that the three SNP loci (16:71634198 G>C, 16:71593153 G>C, 16:71699738 G>C) showed significant differences in genotype frequencies between northern and southern cattle populations (P<0.01), and the candidate genotypes associated with cattle adaptation to cold environments at each locus were all GG.

[0045] (II) Correlation analysis between SNP loci and bovine cold adaptation (1) Processing of temperature and humidity data at the sampling site of the sample population First, the adaptability of different cattle breeds to cold environments was assessed. To this end, the cold and humid conditions of each sampling site were quantified by calculating the annual average temperature (°C) and the "Temperature Humidity Index" (THI).

[0046] Table 4-1. Temperature and humidity levels at different cattle gathering sites

[0047] Table 4-2. Temperature and humidity levels of different cattle gathering sites

[0048] The results are shown in Tables 4-1 and 4-2. The temperature-humidity index comprehensively considers two key climatic factors: air temperature and relative humidity. A lower value indicates a colder and more humid environment. According to the national standard issued by the China Meteorological Administration, the temperature-humidity index can be calculated using the following formula: I= 0.55×(1 )×( 14.4) In the above formula, I represents the temperature and humidity index, which is rounded to one decimal place; Indicates the average annual temperature (°C); This represents the annual average relative humidity. The annual average temperature and relative humidity are calculated based on meteorological data from 1951 to 1980 provided by the National Meteorological Data Network.

[0049] After data processing, the final annual average temperature and temperature-humidity index for each breed will be used as the environmental adaptability phenotypic values ​​for the corresponding breed of cattle.

[0050] (2) ANOVA analysis of the association between genotype frequencies of all cattle samples and annual average temperature (°C) Table 5. LPGAT1 Association analysis between SNPs in gene intron regions and annual average temperature (°C)

[0051] Note: Superscripts for different letters in Table 5 A、B、C The significance level of statistical differences between genotypes is P<0.01; if the superscript letters are the same, there is no significant difference.

[0052] (3) ANOVA analysis of the association between genotype frequencies and temperature and humidity index (THI) in all bovine samples Table 6. LPGAT1 Association analysis between SNPs in gene intron regions and temperature and humidity index (THI)

[0053] Note: Superscripts for different letters in Table 6 A、B、C The significance level of statistical differences between genotypes is P<0.01; if the superscript letters are the same, there is no significant difference.

[0054] The results above (Tables 5 and 6) indicate that the genotype frequencies of the three SNP loci (16:71634198, 16:71593153, and 16:71699738) identified in the Northern Yellow Cattle population are significantly correlated with annual average temperature and temperature-humidity index (THI). Among them, locus 16:71699738 shows the strongest association (highest F value); and each locus exhibits a clear environmental adaptation gradient. The GG, GC, and CC genotypes all show a stepped "ABC" distribution in terms of annual average temperature and temperature-humidity index, with highly significant differences between each pair of genotypes (P<0.01).

[0055] By expanding the range of cattle breeds collected and re-analyzing (the Zaosheng cattle were replaced with Anxi cattle in the analysis; the latter samples were collected in May 2020 from Anxi County, Gansu Province, with an average annual temperature of 1.5℃, an average annual relative humidity of 50%, and a temperature-humidity index of 0.3), the results showed that only two loci, 16:71634198 and 16:71593153, still exhibited a significant gradient in environmental adaptability.

[0056] (iii) Ox LPGAT1 Verification of the biological function of genes (1) Further screening was conducted using multi-omics data, and chromatin accessibility sequencing (ATAC-seq) and histone modification data (including H3K27ac and H3K27me3) were used to functionally validate candidate regions. ATAC-seq is used to detect chromatin accessibility to identify potential regulatory element regions under different environmental conditions; H3K27ac signals represent active enhancer and promoter regions, reflecting the gene transcription activation state; while H3K27me3 is a histone modification associated with transcriptional repression, usually marking silenced gene regions. By integrating data from ATAC-seq, H3K27ac, and H3K27me3 at three omics levels, the selected regions were further functionally validated. LPGAT1 Epigenetic activity analysis of the gene revealed that locus 71634198 on chromosome 16 is located in an open chromatin region, accompanied by enhanced H3K27ac signaling and decreased H3K27me3 signaling. Figure 2 This indicates that the site possesses potential transcriptional activation characteristics, suggesting... LPGAT1Functional role of genes in bovine cold adaptation.

[0057] (2) Transcription factor binding site prediction analysis was performed on SNPs using the JASPAR database. The results showed that at the 71634198 locus on chromosome 16, the reference allele G (i.e., the adaptive allele) of northern cattle showed a higher binding affinity at the KLF15 binding site compared to the mutant allele C of southern cattle. Figure 3 This result suggests that the SNP (16:71634198 G>C) may regulate KLF15 binding by enhancing its binding. LPGAT1 Gene expression plays an important role in bovine cold adaptation.

[0058] (3) Select two representative breeds from both the northern and southern cattle populations, and conduct a comparative analysis. LPGAT1 Gene expression levels were validated, as detailed in Table 7. Multiple tissue samples were collected from Mongolian cattle and Leiqiong cattle for gene expression analysis.

[0059] First, total RNA was extracted from tissues. After passing quality checks, a transcriptome library was constructed, and transcriptome sequencing was performed using the Illumina Hiseq 2500 platform. The obtained raw reads underwent quality control using FASTP to remove adapter sequences and low-quality reads, resulting in high-quality clean reads. The clean reads were then aligned to the bovine reference genome (ARS-UCD1.2) using HISAT2 software. Transcript assembly and quantification were performed using StringTie to obtain... LPGAT1 Gene expression levels (TPM) in each tissue sample were determined. Finally, differential expression analysis of transcripts was performed using the Deseq2 package in R software.

[0060] Analysis results LPGAT1 The gene was differentially expressed in the lung, liver, and spleen. Figure 4 Furthermore, the expression level in northern cattle was significantly higher than that in southern cattle, and it was significantly upregulated. LPGAT1 The expression level of this gene can enhance the thermogenic capacity of brown adipose tissue, thereby improving an individual's adaptability to cold environments. This further suggests... LPGAT1 Genes may play an important regulatory role in cattle's adaptation to cold environments.

[0061] Table 7. Sample Collection Information for Transcriptome Analysis of Yellow Cattle Populations in Northern and Southern China

[0062] The results above indicate that the GG genotype at locus 16:71634198 is an important candidate molecular marker for bovine cold adaptation.

[0063] (iv) Based on LPGAT1 Application of dominant alleles in intron regions in the breeding of cold-adapted yellow cattle First, using inclusion LPGAT1 The reference genome sequence of the gene was selected, and the 16:71634198 site was used as a template by extending a certain length on both sides of the SNP site. The results are shown in Table 8.

[0064] Table 8. Primer Information

[0065] For the individual cattle to be tested, PCR amplification was performed using genomic DNA as a template and the primers described above. After PCR amplification, the size of the amplified products was detected by gel electrophoresis, and genotyping was performed by sequencing. According to the individual genotyping results, if the genotype at locus 16:71634198 is GG, it indicates that the corresponding cattle individual carries a candidate molecular marker and can be selected into herds adapted to cold environments, thereby promoting gene research, genetic improvement, and breeding related to bovine cold adaptation.

Claims

1. A method of detecting a molecular marker associated with cold tolerance in cattle, characterized by: Comprising the following steps: Part of the fragment in the candidate genomic region is amplified by PCR with the bovine genomic DNA as template, and the genotype of the SNP site contained in the amplified fragment is identified; the candidate genomic region is 71.56MB to 71.71MB on the chromosome 16 of bovine reference genome.

2. The method of detecting a molecular marker associated with cold tolerance in cattle according to claim 1, wherein: The SNP site is located at 71634198 on the chromosome 16 of bovine reference genome, the reference allele of the SNP site is G, and the mutant allele is C.

3. A test kit for a molecular marker associated with cold tolerance in cattle, characterized by: The kit comprises primers for amplifying the SNP site located in the candidate genomic region; the candidate genomic region is 71.56MB to 71.71MB on the chromosome 16 of bovine reference genome.

4. The detection kit for molecular markers related to bovine cold adaptation according to claim 3, characterized in that: The SNP site comprises 71634198 on the chromosome 16 of bovine reference genome, and the reference allele of the SNP site is G and the mutant allele is C.

5. Use of a SNP site associated with cold tolerance in cattle in marker assisted selection breeding of cattle, characterized in that: The SNP site is located in the candidate genomic region; the candidate genomic region is 71.56MB to 71.71MB on the chromosome 16 of bovine reference genome.

6. The use of a SNP locus associated with cold tolerance in cattle according to claim 5 in marker assisted selection breeding of cattle, characterized in that: The SNP site is specifically located at 71634198 on the chromosome 16 of bovine reference genome, the reference allele of the SNP site is G, and the mutant allele is C.

7. The use of a SNP locus associated with cold tolerance in cattle according to claim 5 or 6 in marker assisted selection breeding of cattle, characterized in that: The yellow cattle individual with GG genotype is superior to the individuals with GC and CC genotypes in cold tolerance or cold tolerance potential.

8. Use of the method for detecting the molecular marker related to the cold adaptability of bovine in marker-assisted selection breeding of bovine according to any one of claims 1-2.

9. A method for selecting cold-adapted yellow cattle using a single nucleotide genetic marker, characterized by: Comprising the following steps: Part of the fragment in the candidate genomic region is amplified by PCR with the bovine genomic DNA as template, and the genotype of the SNP site contained in the amplified fragment is identified, and then individuals with the genotype related to cold adaptability at the corresponding site are screened from all the bovine to be tested; the candidate genomic region is 71.56MB to 71.71MB on the chromosome 16 of bovine reference genome.

10. The method for breeding cold-adapted yellow cattle using a single nucleotide genetic marker according to claim 9, characterized in that: The SNP site is specifically located at 71634198 on the chromosome 16 of bovine reference genome, the reference allele of the SNP site is G, and the mutant allele is C.

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