Function mutation site combination based on bovine immune disease-resistant tissue regulation region and application

Abstract

The invention belongs to the technical field of animal gene breeding, and particularly discloses a functional mutation site combination based on a bovine immune disease-resistant tissue regulation region and application. According to the chip, the effect of bovine genome mutation sites on the influence of the activity of an immune tissue genome regulation element is fully considered, and 9394 mutation sites (SNP) which highly interfere with the binding strength of bovine transcription factors are screened from a large number of bovine genome mutation sites and immune tissue epigenetic regulation data; each marker provided by the invention is more closely associated with gene expression regulation and phenotype, and cattle disease resistance related character marker-assisted selection or cattle disease resistance breeding can be carried out by adopting a functional mutation site combination based on a cattle immune disease-resistant tissue regulation region.

Description

Technical Field

[0001] This invention belongs to the field of animal gene breeding technology, specifically involving the combination and application of functional mutation sites in the regulatory region of bovine immune disease resistance tissue. Background Technology

[0002] Cattle (Bos taurus) are among the earliest domesticated animals, with their domestication history dating back to the Fertile Crescent approximately 10,000 years ago. As the world's most important economic livestock, cattle play a crucial role in human societal development, providing essential goods such as meat, milk, and leather, and fulfilling vital functions in agricultural production, including plowing and transportation. According to statistics from the Food and Agriculture Organization of the United Nations (FAO, https: / / www.fao.org / livestock-systems / en / ), the global cattle population exceeds 1.5 billion, distributed across almost all agricultural regions, resulting in a rich and diverse breed resource. As a vital global economic livestock, their health and disease resistance directly impact the productivity and sustainable development of animal husbandry. With the promotion of intensive farming, cattle herds face multiple pathogen threats, including bacteria (such as Brucella and Mycobacterium bovis), viruses (such as Foot-and-Mouth Disease Virus (FMDV) and Bovine Viral Diarrhea Virus), and parasites (such as Babesia). These diseases not only cause severe economic losses but also pose a risk of zoonotic diseases. Bovine immune tissue function is closely related to mastitis and pregnancy immune responses in dairy cows, with pregnancy immune responses directly correlated with lactation intervals. Therefore, bovine immune tissue function is closely related to the economic benefits of dairy cows and the healthy development of the dairy industry. Thus, more accurately locating key SNP sites, optimizing disease-resistant breeding strategies, and improving the immune resistance of cattle are important goals of breeding research. Summary of the Invention

[0003] To meet the current needs of cattle genetic breeding and disease resistance improvement in my country, this invention provides a combination of SNP loci based on the regulatory region of bovine immune disease resistance tissues. Utilizing the specific SNP loci provided by this invention, it is possible to efficiently and accurately assess the disease resistance performance of cattle herds, screen for immune traits, and conduct molecular-assisted breeding, which helps improve herd health, optimize germplasm resources, and promote the breeding of new disease-resistant and high-yielding breeds. The detection of this locus is rapid, high-throughput, and low-cost, and has significant application value and market prospects in cattle breeding, disease control, and the selection of high-quality breeding stock.

[0004] To achieve the technical objective of this invention, the following technical solution is provided:

[0005] Screening methods for functional mutation sites in the immune regulatory regions of bovine lymphoid tissue and spleen:

[0006] S1. Identify transcription factor binding sites using epigenetic data on chromatin accessibility and histone modifications from bovine immune tissues, spleen, and lymphoid tissues;

[0007] S2. Using the transcription factor binding site data in S1, the SNP scoring system in the IFmut database was used to perform functional prediction on 70,394 mutation sites in the bovine genome, and candidate functional mutations that highly interfere with the binding strength of bovine transcription factors were screened out.

[0008] S3. Based on existing bovine QTL data, 9394 functional mutation sites were preferentially screened to form a combination of functional mutation sites based on the regulatory region of bovine immune disease resistance tissue.

[0009] Based on the above method, this invention screened 9394 functional mutation sites in the bovine genome that play a role in immune tissues. The combinations of these mutation sites are shown in Table 1. The physical locations of these mutation sites were determined based on the bovine ARS-UCD1.2 reference genome. The tissues in which these mutation sites exert their effects are primarily the bovine immune-related spleen and lymphoid tissues. This set of functional mutation sites has the following uses:

[0010] (1) Application in cattle breeding;

[0011] (2) Application in the analysis of disease resistance traits (disease resistance breeding) in cattle.

[0012] Based on in-depth research on immune-related genes in major domestic and international cattle breeds, this invention provides a combination of key SNP loci for bovine immune traits, consisting of 9394 SNP loci. This SNP locus combination not only possesses strong international applicability and broad coverage, but also enables early assessment of the innate immunity of cattle herds at the gene level, obtaining more precise information for disease-resistant breeding and optimizing the breeding selection process. Utilizing this locus combination, it is possible to screen for bovine immune performance, identify disease-resistant breeds, and trace genetic background, providing strong molecular technical support for improving bovine health, breeding new breeds with high disease resistance, and protecting and improving germplasm resources. Attached Figure Description

[0013] Figure 1 A technical approach for collecting functional mutation sites in the regulatory regions of bovine immune and disease-resistant tissues. Detailed Implementation

[0014] Specific embodiments of the present invention will be further described in the following detailed description. However, it should be noted that these examples are for illustrative purposes only and should not be considered as limiting interpretations of the present invention. Unless otherwise stated, the technical solutions used in the embodiments are all conventional techniques commonly known to those skilled in the art.

[0015] Example 1: Obtaining combinations of bovine immune-related SNP sites

[0016] To meet current needs in bovine disease prevention and control and to reflect the genetic characteristics of bovine immunity worldwide, this invention relies on the genomic data of multiple bovine breeds worldwide to screen and identify functional mutation sites in the regulatory regions of bovine lymphoid tissue and spleen immune tissues. The specific method is as follows:

[0017] (I) Collecting Apparent Data (Bovine Immune Disease Resistance Tissue Regulation Zone)

[0018] 1. Using data on chromatin accessibility and histone modification in bovine spleen and lymphoid tissues, transcription factor binding sites with characteristics such as cell specificity and tissue activity were identified.

[0019] 2. Identification of potential functional mutation sites

[0020] This invention collects potential functional mutation sites in the bovine genome from the comprehensive agricultural animal variation database—IFmut (http: / / www.ifmutants.com:8212 / # / home).

[0021] (II) Identification of immune tissue-specific regulatory regions

[0022] 1. By performing ATAC-sep and ChIP-sep on the data obtained in (I), significantly enriched peaks across the entire genome were identified, which are candidate regulatory regions.

[0023] 2. The intersection of ATAC-seq peaks and ChIP-seq peaks of active markers such as H3K27ac and H3K4me1 is taken and merged to obtain a set of high-confidence "immunotissue activity regulatory regions".

[0024] (III) Identification of transcription factor binding sites

[0025] Within active regulatory regions, Motif analysis and TFBS scanning are performed to precisely locate potential protein-DNA interaction sites, thereby obtaining a list containing TFBS genomic coordinates and their corresponding inferred transcription factor names.

[0026] (iv) Annotation and Screening of Mutations in Regulatory Regions

[0027] We map existing genetic variation data (such as SNPs) onto the regulatory regions and TFBSs of interest, and screen out all SNP sites located in the regulatory regions of immune tissue activity to obtain a list of candidate SNPs located in key regulatory regions and TFBSs.

[0028] (v) Predicting transcription factor binding potential

[0029] Quantification (IV) determines whether the SNPs screened in the quantification process will change the binding affinity of transcription factors, predicts the effect of allele changes on TF binding, obtains the functional prediction score (e.g. deltaSVM score) of each candidate SNP, and sorts them accordingly to screen out the sites with the strongest predictive effect.

[0030] (vi) Colocation Analysis

[0031] Statistical methods (such as COLOC and TWAS) were used to verify whether the functional SNP sites screened in this invention were located in the same genomic region as the known eQTL / GWAS signal sites. Priority was given to those SNP sites that were predicted to disrupt TF binding and were significantly associated with known disease resistance phenotypes. Based on the bovine ARS-UCD 1.2 reference genome, a final 9394 functional mutation sites were generated.

[0032] Table 1 Functional mutation sites in the bovine immune and disease-resistant tissue regulatory region.

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[0083] Example 2: Application of functional mutation sites in the regulatory region of bovine immune disease resistance tissue

[0084] The functional locus set constructed in this invention considers bovine spleen and lymphoid tissue, which are target tissues for immune traits in the body and are closely related to viral infection and autoinflammation. Therefore, the loci of this invention can be used for marker-assisted selection of disease resistance traits (disease resistance breeding). Specific steps:

[0085] (1) Detecting genomic mutation information in cattle using the molecular marker combination of the present invention;

[0086] (2) Construct a population genotype matrix using bovine genome mutation information;

[0087] (3) Use the GBLUP model to construct a bovine genome relationship matrix, conduct association analysis on mastitis, viral resistance (viral load), etc., and select and breed according to the selection index.

[0088] Example 3: Construction of a gene chip for bovine disease resistance breeding

[0089] Using 9394 screened functional mutation sites as core sites, a bovine disease resistance breeding gene chip can be fabricated. Correlation analysis between the chip data and individual health records, immune indicators, etc., can further verify the genetic association between these functional mutations and specific diseases, providing a deeper understanding of the genetic basis of bovine disease resistance. This can be applied to the precision breeding of disease-resistant traits, accelerating the process of selecting superior disease-resistant breeds. Specific steps:

[0090] (1) Using the 9394 target functional mutation sites and their flanking sequences (20 bp before and after each site) selected as targets, a set of specific biotin-labeled double-stranded DNA probe pools were designed and synthesized.

[0091] (2) The fragmented bovine genomic DNA library was hybridized with an excess of biotinylated probes in solution. The probes specifically bound to the target sequences that were completely complementary to the genomic DNA, forming probe-target DNA double-stranded hybrids.

[0092] (3) Add streptavidin-coated magnetic beads to the hybridization reaction system. Magnetic separation is used to remove unhybridized genomic DNA and non-specific sequences, thereby enriching the target region.

[0093] (4) Remove the free probe, amplify the target DNA fragment enriched on the magnetic beads by PCR, and construct a high-quality sequencing library. Sequencing the library and bioinformatics analysis of the sequencing data are performed to obtain the precise genotype of the target functional mutation site.

[0094] Example 4: Application of Bovine Disease Resistance Breeding Gene Chips

[0095] Gene chips prepared using the 9394 functional mutation sites screened above can be used for the following purposes:

[0096] (1) Utilize gene chips to accurately predict disease resistance breeding values ​​in the early stages and accelerate the process of genetic improvement;

[0097] (2) Optimize the selection and breeding process to avoid mating of individuals with the same genetic defects and actively combine superior disease-resistant genes for breeding.

[0098] (3) Using gene chips to screen out local superior individuals carrying disease-resistant genes is beneficial to the protection of local germplasm resources;

[0099] (4) Using chips for disease resistance grading is beneficial for large-scale breeding enterprises to carry out refined management, improve the health level of cattle, and thus improve reproductive efficiency.

[0100] (5) Improve the safety and quality of beef and dairy products.

[0101] The reproductive capacity of dairy cows directly impacts the economic benefits of farms, and the milking interval is a reflection of this capacity. The milking interval is inextricably linked to the cow's immune system; various diseases can affect it, including infectious diseases such as brucellosis and bluetongue, nutritional and metabolic diseases such as milk fever and ketosis, and common dairy cow diseases such as mastitis and reproductive organ diseases. Therefore, disease-resistant breeding gene chips can be used to assist in predicting this complex trait of milking interval, thereby selecting superior breeds, accelerating the genetic improvement process, and increasing dairy cow reproductive efficiency.

[0102] To illustrate the application value of the functional locus gene chip obtained in this invention, the breeding value (EBV) of the complex trait of milking interval in dairy cows was estimated using whole-genome mutation sites from 400 dairy cows and functional mutation sites identified in this invention. The accuracy of the two estimated breeding values ​​(EBV) was compared. The estimation was repeated five times, with 20% of all individuals selected as the prediction group each time. The correlation coefficient between the EBV of the prediction group and the actual phenotype of the prediction group was used as the EBV estimation accuracy. The results showed that the functional locus gene chip based on this invention had higher accuracy in estimating the EBV of this trait, demonstrating the good effect of functional locus gene chips in accurately predicting complex traits at an early stage and accelerating the genetic improvement process. It also demonstrated the potential of functional mutations screened based on functional locus gene chips in elucidating the genetic mechanisms of complex traits to accelerate downstream industrial applications.

[0103] Table 2. Comparison of the accuracy of breeding values ​​for the milk production interval trait in dairy cows between traditional whole-genome chips and functional site-specific gene chips.

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Claims

1. The application of SNP locus combinations in marker-assisted selection of bovine disease resistance traits or in bovine disease resistance breeding, characterized in that, The information of the SNP sites is shown in Table 1 of the specification. The physical location of the mutation sites is determined based on the bovine ARS-UCD1.2 reference genome.

2. A molecular probe assembly, characterized in that, The molecular probe combination detects the functional mutation sites shown in Table 1 of the instruction manual, the physical location of which is determined based on the bovine ARS-UCD1.2 reference genome.

3. A gene chip based on functional mutation sites in the regulatory region of bovine immune disease-resistant tissues, characterized in that, The chip is loaded with the molecular probe assembly as described in claim 2.

4. The application of the molecular probe combination described in claim 2 in marker-assisted selection of bovine disease resistance traits or in bovine disease resistance breeding.

5. The application of the gene chip according to claim 3 in marker-assisted selection of bovine disease resistance traits or in bovine disease resistance breeding.

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