Method for breeding and improving concentration of cattle fibrinogen through SNP (Single Nucleotide Polymorphism) marker and application of method

Abstract

The invention relates to a method for breeding and improving the fibrinogen concentration of Tibetan cattle through SNP markers and application of the method. The invention provides an SNP (Single Nucleotide Polymorphism) marker for detecting the fibrinogen concentration of the improved Tibetan cattle, and if the detected mononucleotide at the SNP marker is T, the fibrinogen concentration of the improved Tibetan cattle individual is relatively high; if the mononucleotide at the SNP marker is detected to be C, the fibrinogen concentration of the improved Tibetan cattle individual is relatively low. The improved Tibetan cattle are screened according to the detection result, the improved Tibetan cattle individuals capable of adapting to the plateau low-oxygen environment are screened, the survival and adaptive capacity of the improved Tibetan cattle in the plateau area is improved, and powerful support is provided for genetic breeding of the improved Tibetan cattle and development of dairy cows in the plateau area.

Description

Technical Field

[0001] This application belongs to the fields of molecular biotechnology and SNP marker technology, and relates to a method for selecting fibrinogen from cattle by SNP marker and its application. Background Technology

[0002] Holstein-Friesian cattle, originating in the Netherlands and northern Germany, are the world's most common high-yielding dairy cattle breed. These cattle are renowned for their distinctive black and white spots and excellent milk quality, exhibiting high milk production and favorable genetic traits (Xinyu Zhang, 2022). However, in specific environments, such as the low-oxygen and low-temperature conditions of the Tibetan Plateau, Holstein cattle struggle to adapt because their large size and high metabolic demands are easily limited at high altitudes. On the other hand, Tibetan cattle, a native dairy breed originating from the Qinghai-Tibet Plateau, are particularly adapted to the low-oxygen and cold environment of the plateau. Although their milk production is lower, they possess excellent cold resistance and a high capacity for digesting roughage (Deng F, 2015). Considering the characteristics of these two breeds, the improved Tibetan cattle bred through crossbreeding Holstein males and Tibetan females can adapt to the unique climate of the plateau while inheriting the high milk production and excellent milk quality of Holstein cattle. This crossbreeding strategy not only holds promise for improving dairy farming efficiency in plateau regions but also contributes to enhancing the economic benefits of the local dairy industry. However, with the increase in the number of generations of improved Tibetan cattle, the incidence of altitude sickness has increased, seriously affecting the quality of milk and the development of the local dairy industry. In Shannan and Shigatse areas of Tibet, most of the local improved cattle (Holstein males × Tibetan females) have entered the fifth generation (F5), and some have even entered the sixth generation (F6). After the third generation (F3), some traits that are not conducive to production have gradually become prominent, such as declining reproductive performance, a sharp increase in the mortality rate of cows due to dystocia, a calf birth mortality rate of about 5%, and a newborn calf survival rate of less than 90%. During the feeding process, they are prone to edema, high altitude polycythemia, and other phenomena. Their poor adaptability to high altitude seriously affects the yield and quality of dairy products and has also had a huge impact on local animal husbandry. Solving the problem of the adaptability of improved cattle is now urgent.

[0003] In high-altitude environments, animals face multiple challenges, including oxygen scarcity, intense ultraviolet radiation, and low temperatures (Wu et al., 2018), which can lead to tissue damage or inflammation. In these conditions, the role of fibrinogen becomes particularly significant. It helps form blood clots, reduces the risk of bleeding, and promotes the repair of damaged tissues. Therefore, fibrinogen plays a crucial role in high-altitude adaptation, assisting animals in better adapting to the survival challenges of high-altitude environments and maintaining bodily integrity to more effectively adapt to hypoxic conditions. This highlights the vital importance of fibrinogen in maintaining animal health and survival.

[0004] Fibrinogen is a plasma protein synthesized by the liver. As a precursor protein of fibrin, it is encoded by different genes, but they are structurally very similar, ensuring their functional consistency. It plays a crucial role in the body. Fibrinogen plays a key role in tissue injury and participates in the coagulation process. In the coagulation cascade, fibrinogen is first converted into soluble fibrin, and then further polymerizes into insoluble fibrin, which helps to form a thrombus to control bleeding. In addition, fibrinogen also plays multiple roles in tissue damage repair and inflammation (Luyendyk JP et al., 2019; Donkin R et al., 2023; Liu et al., 2017).

[0005] Against this technological backdrop, single nucleotide polymorphism (SNP) markers are widely used in genetic research. They can locate variations at specific sites in the genome and establish associations with physiological characteristics. Therefore, SNP marker-assisted breeding has become a powerful option for improving fibrinogen concentration in improved Tibetan cattle. Overall, modern genetic methods such as SNP marker-assisted breeding and genome-wide association studies will provide more accurate tools and methods for improving fibrinogen concentration in improved Tibetan cattle, enhancing their survival and growth capabilities under different environmental conditions, and providing the livestock industry with more competitive production animals.

[0006] The study of SNP markers related to fibrinogen in improved Tibetan cattle (Holstein male × Tibetan female) has broad application prospects. It not only helps to scientifically understand the adaptation mechanism, but also provides solid support for the genetic improvement of Tibetan cattle and animal husbandry in plateau areas. Summary of the Invention

[0007] The purpose of this application is to explore a method for selecting fibrinogen concentration in cattle by SNP markers and its application.

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

[0009] The first aspect of this application provides an SNP marker for detecting the concentration of fibrinogen in modified Tibetan cattle, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0010] Furthermore, the single nucleotide M position of the SNP marker is located at the T>C mutation at nucleotide position 50683493 on chromosome 16 of the Bos taurus reference genome ARS-UCD1.2 version, and its position is at position 11 at the 5' end in sequence SEQ ID NO: 1.

[0011] The second aspect of this application provides a primer pair for identifying SNP markers of fibrinogen concentration in modified Tibetan cattle, the primer pair comprising an upstream primer and a downstream primer;

[0012] The nucleotide sequence of the upstream primer is: 5'-GGTCCATTATCTGCTCACT-3'; the nucleotide sequence of the downstream primer is: 5'-GGCTGCTGTCCTTATCAT-3'.

[0013] The third aspect of this application provides a method for detecting the concentration of fibrinogen in improved Tibetan cattle, the specific steps of which include: using an SNP marker with a nucleotide sequence as shown in SEQ ID NO: 1, detecting the single nucleotide of the SNP marker to obtain a detection result; used to determine the level of fibrinogen concentration in the improved Tibetan cattle, wherein the position of the single nucleotide M of the SNP marker is located at the T>C mutation at nucleotide site 50683493 on chromosome 16 of the Bostaurus reference genome ARS-UCD1.2 version, and its position is at position 11 at the 5' end in the sequence SEQ ID NO: 1.

[0014] Furthermore, the method also includes: when the detection result is that the single nucleotide M is T, it is determined that the improved Tibetan cattle individual has a high fibrinogen concentration and is a TT type individual; when the detection result is that the single nucleotide M is C, it is determined that the improved Tibetan cattle individual has a low fibrinogen concentration and is a CC type or TC type individual.

[0015] The fourth aspect of this application provides a method for selecting and improving Tibetan cattle individuals, the specific steps of which include:

[0016] 1) Extract genes from the individual Tibetan cattle to be tested for improvement;

[0017] 2) Detect nucleotide sites on chromosome 16 of improved Tibetan cattle. Among the SNP-labeled single nucleotides, retain improved Tibetan cattle individuals with M-labeled single nucleotides of T, and eliminate improved Tibetan cattle individuals with M-labeled single nucleotides of C.

[0018] 3) Increase the frequency of the homozygous TT genotype at this nucleotide site generation by generation, thereby increasing the fibrinogen concentration in the offspring of improved Tibetan cattle.

[0019] The fifth aspect of this application provides the application of an SNP marker for detecting the fibrinogen concentration in improved Tibetan cattle in the selection and breeding of improved Tibetan cattle.

[0020] The sixth aspect of this application provides a method for selecting and breeding improved Tibetan cattle individuals, and its application in improving the fibrinogen concentration of improved Tibetan cattle.

[0021] Beneficial effects

[0022] 1. This application provides an SNP marker related to fibrinogen in improved Tibetan cattle, and also provides a primer pair for identifying SNP markers that affect fibrinogen in improved Tibetan cattle. Ultimately, it establishes an efficient and accurate SNP marker-assisted breeding program for improved Tibetan cattle, which can be applied to the genetic improvement of fibrinogen concentration regulation in improved Tibetan cattle. This can appropriately increase the fibrinogen concentration in improved Tibetan cattle in the plateau environment, which helps to promote the repair of blood vessels and tissues in improved Tibetan cattle in the plateau, improve inflammation and immune regulation, and thus enhance the adaptability of improved Tibetan cattle to the plateau environment of low oxygen, strong ultraviolet radiation and low temperature.

[0023] 2. This application, by selecting the dominant allele of the SNP marker, can improve the genetic progression of fibrinogen concentration in improved Tibetan cattle, thereby effectively improving the breeding efficiency of improved Tibetan cattle. In particular, through this SNP marker, this application can select all CC and CT individuals into TT individuals, which helps to promote the repair of blood vessels and tissues in improved Tibetan cattle in the plateau, improve inflammation and immune regulation, thereby enhancing the adaptability of improved Tibetan cattle to the plateau environment of low oxygen, strong ultraviolet radiation and low temperature, which is beneficial to the growth and development of improved Tibetan cattle in the plateau.

[0024] 3. This application provides a SNP marker associated with fibrinogen in improved Tibetan cattle, enabling more precise study of genetic factors related to fibrinogen concentration in improved Tibetan cattle through genome-wide association analysis (GWAS). This SNP marker can address the following issues:

[0025] 1) Improved accuracy: A SNP marker associated with fibrinogen in improved Tibetan cattle is provided, which can more accurately locate fibrinogen-related genes, rather than relying solely on randomly selected candidate genes.

[0026] 2) Reduce uncertainty: A SNP marker associated with fibrinogen in improved Tibetan cattle is provided to reduce uncertainty. The correlation between the SNP marker and fibrinogen in the directly associated genome is obtained through genome-wide association analysis (GWAS). By selecting the dominant allele of this SNP marker, the genetic progression of fibrinogen in improved Tibetan cattle can be improved, thereby improving the survival and growth ability of improved Tibetan cattle in plateau areas and thus improving the economic benefits of improved Tibetan cattle breeding in high-altitude areas.

[0027] 3) Providing precise tools: The SNP marker provided in this application is a T>C nucleotide mutation located at position 11 of the sequence marked in SEQ ID NO:1; the SNP marker site is located at nucleotide position 50683493 on chromosome 16 of the Bos taurus reference genome ARS-UCD1.2, which will provide a more accurate genetic tool for improving the survival and growth ability of improved Tibetan cattle in plateau areas, and provide a theoretical basis for improving the survival and growth ability of improved Tibetan cattle in plateau areas. Attached Figure Description

[0028] Figure 1 The M in the sequence listing is the mutation site, indicated by bold M. The positions of the primer sequences are indicated by bolding at the beginning and end of the sequence.

[0029] Figure 2 This is a Manhattan diagram related to fibrinogen (Manhattan diagram of fibrinogen GWAS association analysis).

[0030] Figure 3 It is a QQ image related to fibrinogen.

[0031] Figure 4 This is a Manhattan plot related to fibrinogen (MANGYN plot of fibrinogen GWAS association analysis).

[0032] Figure 5 The effect of different genotypes at the g.11 locus on HGB (2500m).

[0033] Figure 6 The effect of different genotypes at the g.11 locus on HGB (3500m).

[0034] Figure 7 This is a distribution map of sequencing sites.

[0035] Figure 8 This is a statistical chart of the sample sequencing data results.

[0036] The horizontal axis represents the sample name, and the vertical axis represents the percentage. NA_rate ≤ 10% indicates that the sample detection missing rate meets the requirements. The lower the NA_rate, the fewer undetected sites the sample has. Het_alt_rate, Hom_alt_rate, and Ref_rate are sample-related.

[0037] Figure 9 This is a flowchart illustrating the working principle of GenoBaits.

[0038] Figure 10 Primer verification was performed using the UCSC database. Specific implementation plan

[0039] The present application will be described in further detail below with reference to the embodiments and accompanying drawings, but the implementation of the present application is not limited thereto.

[0040] The above-mentioned inventive objective of this application is achieved in the following specific way:

[0041] experimental animals

[0042] The experimental improved Tibetan cattle (Holstein male × Tibetan female) used in this application were all from 2258 F3 generation improved Tibetan cattle from Lhasa, Shannan, and Shigatse in the Tibet Autonomous Region. The cattle were aged 4-5 years and were raised in improved Tibetan cattle farms with similar environmental conditions, including similar altitudes and climates. The herd pedigree records are available in Table 1 below. The improved Tibetan cattle had free access to grazing and water, and the feeding methods and conditions remained consistent throughout the process, following conventional feeding and management practices.

[0043] Table 1. Sample Information for Genotyping Detection

[0044]

[0045] Sample collection

[0046] A total of 2258 F3 generation Tibetan cattle (4-5 years old) from improved Tibetan cattle breeds in Lhasa, Shannan, and Shigatse, Tibet Autonomous Region, were collected and raised in similar environments with similar altitudes and climates. Blood samples were collected from the jugular vein of these cattle and stored at low temperature in a foam box with ice packs. All samples were analyzed for fibrinogen using a BC-2 800Vet fully automated veterinary hematology analyzer within 4 hours. Fibrinogen-detected samples were stored at -20°C for subsequent whole-genome DNA extraction.

[0047] Example 1: Improved Tibetan Yellow Cattle 10K Liquid Chromatography Chip SNP Genotyping

[0048] A 10k liquid-phase microarray for cattle: Whole-genome sequencing was performed on cattle populations at continuous altitudes to identify altitude-associated loci related to hypoxia adaptation. Genome-wide association analysis was also conducted on third-generation individuals of improved Tibetan Yellow cattle (Holstein male × Tibetan Yellow female) to link blood physiological and hemorheological parameters with high-altitude adaptation SNP loci. A liquid-phase microarray containing a set of 11,637 SNP loci was preliminarily designed and developed, aiming to provide an efficient and reliable technical means for conducting genetic breeding work on stress resistance traits in cattle. This approach is convenient, rapid, and low-cost, reducing the problem of long generation intervals during selection and bringing greater economic benefits to farms.

[0049] The 10K liquid-phase chip for cattle plateau adaptation breeding is achieved through the following technical solution:

[0050] Sample collection: Samples of Tibetan cattle and local Yunnan cattle were collected at different altitudes. 5 ml of blood was extracted using vacuum blood collection tubes containing EDTA anticoagulant for whole-genome resequencing of DNA.

[0051] DNA extraction and detection: DNA was extracted using a blood genomic DNA extraction kit, and the purity and integrity of the DNA were detected using 1% agarose gel electrophoresis.

[0052] Whole-genome resequencing: Qualified DNA samples are fragmented into 300-500 bp fragments, followed by end modification and sequencing adapter addition. Products of suitable length are selected by gel electrophoresis, and sequencing libraries are prepared by PCR amplification. Paired-end sequencing is performed using the Illumina HiSeq2500 platform.

[0053] SNP calling and sequence alignment: Quality control of sequencing data is performed, BWA software is used to align to the reference genome, and GATK software is used to perform SNP calling to obtain a high-quality set of gene variation information.

[0054] Population structure and kinship assessment: Linkage disequilibrium filtering was performed on SNP data, principal component analysis was conducted using EIGENSOFT, population structure analysis was performed using ADMIXTURE software, IBS distance between individuals was calculated, a neighbor tree was constructed, and kinship between individuals was explored.

[0055] Locus selection methods: Natural selection was assessed based on the degree of differentiation among populations, and the FST index was calculated using VCFtools. Allele frequency and altitude association analysis was conducted using both MEMEA and BayPass methods to screen SNP loci that were highly associated with trends in environmental factor changes.

[0056] Chip design and development: The SNP sites obtained through the above analysis will be optimized and screened, probes will be designed and synthesized, capture sites will be determined, and chip performance will be tested to ensure high detection rate and accuracy.

[0057] Chip detection: DNA is extracted from the cattle to be tested, a library is constructed, and sequencing is performed to obtain the genotype of the target SNP, which is then applied to genotype detection, genome-wide association analysis, and genome-wide selection breeding of cattle.

[0058] In developing a 10K liquid-phase chip for cattle, we selected approximately 10,000 foreground markers that provided us with important genetic information, which critically points to high-altitude adaptation traits.

[0059] To ensure broad coverage of genetic variation and improve the representativeness of the microarray, we selected 30 cattle with high genetic diversity for 10-fold coverage resequencing. This allowed us to capture more genetic variations, thereby improving the microarray's usability and accuracy. Next, applying the Boreddy SNP marker selection rules, we carefully selected 5,000 to 8,000 background markers evenly distributed on the chromosomes of the improved Tibetan Yellow Cattle.

[0060] Finally, by combining foreground and background markers, we successfully developed an improved Tibetan cattle liquid chromatography-mass spectrometry (LC-MS) chip containing a set of 11,637 SNP loci. This chip represents a significant advancement in plateau cattle breeding, providing a powerful molecular tool for the precise selection of cattle highly adaptable to the plateau environment, greatly improving the efficiency of breed improvement and selection. (Test results of the improved Tibetan cattle 10K L-MS chip sample are shown in Table 2.)

[0061] Table 2. Test results of the improved Tibetan cattle 10K liquid phase chip sample.

[0062]

[0063] Example 2: Detection of SNP markers

[0064] DNA Sample Extraction and Detection: We used a high-throughput DNA extraction kit to extract genomic DNA from the samples. The extracted DNA samples underwent two tests: (1) DNA purity and integrity were analyzed using 1% agarose gel electrophoresis; (2) DNA concentration was accurately quantified using Qubit. (The reagents used in the experiment are shown in Table 3 below).

[0065] Table 3. Reagents used in DNA sample extraction and detection experiments.

[0066]

[0067] GenoBaits experimental procedure: Quantitatively qualified DNA is randomly physically broken using an ultrasonic disruptor, with fragment peaks controlled at 200-300 bp. After end repair, the broken DNA is ligated with A-tails. The A-tailed DNA fragments are ligated to sequencing adapters using ligase. Then, the library is purified and fragments are selected using carboxyl-modified magnetic beads, retaining ligation products with insert fragments of 200-300 bp. The ligation products are then added to barcoded sequencing primers and a high-fidelity PCR reaction system for PCR amplification. Different barcodes are used to distinguish different samples. After purification with carboxyl-modified magnetic beads, the amplified products are ready for probe hybridization experiments.

[0068] Take 500 ng of the constructed sequencing library, lyophilize it, add probes and hybridization reagents, denature it, and incubate at 65°C for 2 hours to complete the hybridization reaction. After washing the hybridization product with washing buffer, perform another round of PCR to complete the construction of the hybridization capture library. Add multiplex PCR panel mix and multiplex PCR amplification enzyme system to the qualified DNA and perform PCR reaction on a PCR instrument. After purifying the PCR product with carboxyl magnetic beads, add it again to the sequencing primers with barcodes and high-fidelity PCR reaction system for PCR amplification. Different barcodes are used to distinguish different samples. After amplification of the product after purification with carboxyl magnetic beads, the multiplex PCR capture and library construction are completed (see GenoBaits working principle and process for details). Figure 9 ).

[0069] Library construction and sequencing: Samples that passed the DNA quality control were used to construct targeted sequencing libraries using appropriate products, thus completing the library preparation for all samples in this project. After library construction, preliminary quantification was performed using Qubit 2.0, followed by accurate quantification of the effective library concentration using qPCR to ensure library quality. Once the libraries passed the quality control, they proceeded to the sequencing stage.

[0070] DNA samples were sent to Borui Di (Shijiazhuang) Biotechnology Co., Ltd. for SNP genotyping and genotyping of modified Tibetan cattle using a 10K liquid chromatography-mass spectrometry (LC-MS) chip on the Illumina Beadstration platform, following the company's standard procedures. The checkmarker function from the GenABEL package in R was used for quality control of the 10K chip scanning genotyping data for all samples, removing samples with a detection rate below 90%, a family Mendelian error rate above 0.1, a minimum allele frequency below 0.05, and a Hardy-Weinberg equilibrium significance level above 10. -6 The SNPs were analyzed, and finally, 10,180 effective genotype data were obtained (see the statistical results of the effective genotype data). Figure 7 , Figure 8 ).

[0071] Example 3: Genome-wide association study (GWAS)

[0072] Currently, a series of potential genes and QTLs related to fibrinogen have been successfully identified using candidate gene methods and QTL mapping. These potential genes include FGB, FGA, FGG, and F2, which are closely related to the structure and function of fibrinogen. Furthermore, we have observed a gradual increase in fibrinogen-related QTLs, which can influence the genetic diversity of fibrinogen. While these methods contribute to our understanding of the genetic basis of fibrinogen, they also have some limitations.

[0073] First, the candidate gene method has limitations. The selection of potential genes is relatively random, meaning that the selected genes may include major effector genes as well as genes closely linked to QTLs but indirectly affecting traits. This presents a challenge for livestock breeding. Furthermore, the candidate gene method is susceptible to interference from population heterogeneity, primarily manifested in reduced breeding accuracy, decreased generalization of restriction effects, increased variability in selection responses, and potential genetic drift and inbreeding. This implies that when applying marker-based breeding techniques in populations with high genetic diversity, additional considerations and strategies may be needed to ensure the universality and effectiveness of the selected markers, thereby avoiding inefficient selection or unpredictable genetic consequences.

[0074] Secondly, QTL mapping typically yields a broad range of QTLs, potentially encompassing hundreds of genes, which limits its application in improving important economic traits in livestock. This also makes the precise screening of potential genes associated with specific traits more challenging.

[0075] Therefore, genome-wide association studies (GWAS) have become a powerful tool for addressing the aforementioned issues. GWAS and SNP markers are interdependent. SNP markers provide genetic polymorphism information for association analysis, while GWAS utilizes this information to reveal the genetic basis associated with phenotypic traits. GWAS involves screening for SNP markers across the entire genome, scanning the studied subjects, and analyzing the association between the obtained SNP marker data and phenotypic traits. Compared to candidate gene methods and QTL mapping, GWAS can more accurately locate and identify genes associated with specific traits, providing a more precise and feasible method for identifying genes related to fibrinogen in improved Tibetan cattle. Against this backdrop, researchers are increasingly focusing on using SNP marker methods to study genetic factors related to fibrinogen. The genetic characteristics of fibrinogen are a complex issue, influenced by multiple genes and environmental factors. A comprehensive understanding and prediction of its performance requires consideration of various factors, including environmental and gene-environment interactions. Traditional breeding methods have limited effectiveness in improving this trait, primarily relying on phenotypic selection, such as excluding individuals with fibrinogen abnormalities or avoiding their offspring for breeding stock. These methods typically fail to achieve ideal breeding results. Therefore, SNP marker-assisted breeding has become a powerful option for improving fibrinogen concentration. This will provide more precise tools and methods for improving fibrinogen concentration in improved Tibetan cattle. This is expected to enhance the survival and growth capabilities of improved Tibetan cattle under different environmental conditions, while also providing the livestock industry with more competitive production animals.

[0076] To eliminate population stratification effects, this application employs a linear mixed model with single-point regression analysis combined with Tassel 5 software for GWAS analysis. The stratification effect is corrected for using inter-individual genomic similarity in the analysis model. The Bonferrin i method is used to determine the significance threshold for the association between SNPs and the effective total number of loci. The genomic significance threshold is 0.05 divided by the number of effective SNP loci, i.e., the genomic significance threshold is 4.91 × 10⁻⁶. -6 That is, 0.05 / 10180 (number of effective SNPs); the significance threshold at the chromosome level is 1 divided by the number of effective SNPs, that is, the significance threshold at the chromosome level is 9.82 × 10 -5 That is, 1 / 10180 (number of valid SNPs).

[0077] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It was found that a site significantly affecting fibrinogen concentration exists on chromosome 16 in the experimentally improved Tibetan cattle herd. The T>C mutation is located at nucleotide 50683493 on chromosome 16 of the Bos taurus reference genome ARS-UCD1.2 version. Its position is at position 11 at the 5' end in sequence SEQ ID NO: 1. The most strongly associated SNP site is g.11T>C (P = 7.12e-07).

[0078] Example 4: Association analysis between different genotypes and fibrinogen

[0079] The fibrinogen concentration of 582 improved Tibetan cattle at medium altitude (2500m) and 2258 at high altitude (3500m) was measured. The fibrinogen content of the three genotypes was analyzed by one-way analysis using SPSS software (the results are shown in Tables 4 and 5).

[0080] As shown in Tables 4 and 5, the SNP site g.11T>C of the SNP marker was significantly correlated with fibrinogen (P<0.001), indicating that this SNP marker significantly affects the fibrinogen of improved Tibetan cattle. Assisted selection at this SNP site in improved Tibetan cattle can improve the fibrinogen of this population, which is beneficial for improving the adaptation of Tibetan cattle to the low oxygen partial pressure, strong ultraviolet radiation, and low temperature environment in high-altitude areas.

[0081] Furthermore, Tables 4 and 5 show that the CC genotype has lower fibrinogen levels than the TT and TC genotypes, indicating that the homozygous TT genotype promotes fibrinogen secretion. For improved Tibetan cattle raised at high altitudes, the animals face multiple challenges, including oxygen scarcity, strong ultraviolet radiation, and low temperatures, which can lead to tissue damage or inflammation. In this context, the role of fibrinogen becomes particularly significant. It helps form blood clots, reduces the risk of bleeding, and promotes the repair of damaged tissues. Therefore, fibrinogen plays a crucial role in high-altitude adaptation, assisting animals in better adapting to the survival challenges of the high-altitude environment and maintaining bodily integrity to more effectively adapt to hypoxic conditions. Therefore, eliminating the CC genotype in improved Tibetan cattle can bring greater economic benefits. In the breeding process, we need to eliminate CC and CT genotypes in improved Tibetan cattle breeding, while retaining the TT genotype, to gradually increase the frequency of the T allele at this locus.

[0082] Table 4. Results of single-factor analysis of fibrinogen from three genotypes (3500m improved Tibetan cattle)

[0083]

[0084] Table 5. Results of single-factor analysis of fibrinogen from three genotypes (2500m improved Tibetan cattle).

[0085]

[0086] Example 5: Invention process for detecting SNP markers

[0087] Primer design: The target fragment containing the SNP site significantly associated with the modified Tibetan cattle fibrinogen was a 220 bp nucleotide sequence from chromosome 16. Primers were designed using Primer Premier 6.0 software. The upstream and downstream primer sequences for sequence amplification are as follows:

[0088] Upstream primer (SEQ ID NO: 2): 5'-GGTCCATTATCTGCTCACT-3'

[0089] Downstream primer (SEQ ID NO: 3): 5'-GGCTGCTGTCCTTATCAT-3'

[0090] PCR amplification: Add 1 μL of DNA template, 3.4 μL of double-distilled water, 5 μL of 2×Tag PCR StanMixwith Loading Dye, and 0.3 μL each of the upstream and downstream primers to a 10 μL reaction mixture. PCR conditions were: 94℃ pre-denaturation for 2 min, followed by 35 cycles of 94℃ denaturation for 30 s, 55℃ annealing for 20 s, and 72℃ extension for 30 s, with a final extension at 72℃ for 10 min. Primers were validated using the UCSC database. Figure 10 ).

[0091] DNA sequencing: Finally, the PCR amplification products were sequenced. The sequencing was performed by BGI Genomics. The gene fragment sequencing was required to be bidirectional. The sequenced sequences were compared with the NCBI genome sequence to determine the mutations at the corresponding SNP sites.

[0092] Effect analysis of SNP-labeled g.11T>C sites on fibrinogen concentration

[0093] This application provides a SNP marker that can significantly increase fibrinogen in improved Tibetan cattle. Using this SNP for marker-assisted selection can greatly accelerate the breeding process for fibrinogen selection in improved Tibetan cattle. If this application successfully breeds all CC-type individuals with the SNP marker affecting fibrinogen in improved Tibetan cattle into TT-type individuals, the fibrinogen secretion of the improved Tibetan cattle will be moderately enhanced. High-altitude animals face environmental factors such as oxygen scarcity, strong ultraviolet radiation, and low temperatures, which may lead to tissue damage or inflammation. In this situation, the role of fibrinogen becomes particularly significant. It helps form thrombi, reduces the risk of bleeding, and promotes the repair of damaged tissues. By selecting the dominant allele (T) of this SNP in improved Tibetan cattle within the individuals with this SNP marker, the economic benefits of improved Tibetan cattle can ultimately be improved, thereby enhancing the economic benefits for herders.

[0094] This application utilizes the detection of the 11th base mutation site in the SEQ ID NO: 1 sequence to conduct preliminary association analysis between its genotype and the fibrinogen of improved Tibetan cattle, providing a new SNP marker for SNP marker-assisted selection of improved Tibetan cattle.

[0095] The above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

[0096] SEQ ID NO: 1 is:

[0097] GGTGG AGCCC T(M)GGCT GCCAC CTGGG CACTT GCCTG ATTCC CAGCC GCACA CATGCAGCCC ATACC TACCA AGGGA GGCCA CTCCACTCCA CCCCT AGGCT GGAGT CAGGT CCCTA GAAAGAGGCC CCAGGGCTGG CAGAA GTGGC CGATG GGGCT CCAGT CACAT TGTGG GGAGTGTGGC CGG

[0098] SEQ ID NO: 2 is:

[0099] GGTCCATTATCTGCTCACT

[0100] SEQ ID NO: 3 is: <^

[0101] GGCTGCTGTCCTTATCAT

Claims

1. A SNP marker for detecting fibrinogen concentration in modified Tibetan cattle, characterized in that, The nucleotide sequence of the SNP marker is shown in SEQ ID NO:

1.

2. The SNP marker according to claim 1, characterized in that, The single nucleotide M position of the SNP marker is located at the T>C mutation at nucleotide position 50683493 on chromosome 16 of the Bostaurus reference genome ARS-UCD1.2 version, and its position is the 11th position at the 5' end in the sequence SEQ ID NO:

1.

3. A primer pair for identifying SNP-labeled fibrinogen concentration in modified Tibetan cattle, characterized in that, The SNP-tagged nucleotide sequence is shown in SEQ ID NO: 1, and the primer pair includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

4. A method for detecting the concentration of fibrinogen in modified Tibetan cattle, characterized in that, The method includes: using an SNP marker with a nucleotide sequence as shown in SEQ ID NO: 1, detecting the single nucleotide of the SNP marker to obtain a detection result, which is used to determine the concentration of fibrinogen in the improved Tibetan cattle. The single nucleotide M of the SNP marker is located at the T>C mutation at nucleotide site 50683493 on chromosome 16 of the Bostaurus reference genome ARS-UCD1.2 version, and its position is at position 11 at the 5' end in the sequence SEQ ID NO:

1.

5. The method according to claim 4, characterized in that, The method further includes: when the detection result is that the single nucleotide M is T, it is determined that the improved Tibetan cattle has a high fibrinogen concentration and is a TT-type individual; when the detection result is that the single nucleotide M is C, it is determined that the improved Tibetan cattle has a low fibrinogen concentration and is a CC-type or TC-type individual.

6. A method for selecting and improving Tibetan cattle individuals, characterized in that, The method includes: 1) Extract genes from the individual Tibetan cattle to be tested for improvement; 2) Detect nucleotide sites on chromosome 16 of improved Tibetan cattle. Among the SNP-labeled single nucleotides, retain improved Tibetan cattle individuals with M-labeled single nucleotides of T, and eliminate improved Tibetan cattle individuals with M-labeled single nucleotides of C. 3) Increase the frequency of the homozygous TT genotype at this nucleotide site generation by generation, thereby increasing the fibrinogen concentration in the offspring of improved Tibetan cattle.

7. The application of the SNP marker as described in claim 1 or 2 in the selection and improvement of fibrinogen concentration in Tibetan cattle.

8. The application of the method for breeding improved Tibetan cattle individuals as described in claim 6 in improving the fibrinogen concentration of improved Tibetan cattle.

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