Gene related to cow mastitis resistance, molecular marker and application

Abstract

The invention discloses a gene related to cow mastitis resistance, a molecular marker and application, and belongs to the technical field of genetic breeding. The genes related to the resistance of the dairy cow mastitis comprise genes HK1, POLR3C, RSAD2 and LRIG2. According to the invention, a selection signal of significant allele frequency difference between an anti-mastitis dairy cow and a mastitis susceptible dairy cow population is searched by utilizing a genome sequence of the dairy cow and a whole genome re-sequencing means, and a gene related to the mastitis resistance and a corresponding SNP molecular marker are screened out and can be used for identifying the dairy cow with high mastitis resistance character; the efficiency of molecular design breeding is improved, and favorable genotype individuals can be selected for breeding, so that the mastitis resistance character of a dairy cow group is improved, the mastitis resistance of the dairy cow group is improved, the treatment cost is reduced, and the economic benefit of a pasture is improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to a gene and a molecular marker related to cow mastitis resistance and an application thereof. Background Art

[0002] Cow mastitis is a common inflammatory disease of the breast of dairy cows, usually caused by infection with a variety of pathogenic microorganisms, such as Staphylococcus aureus, Streptococcus, Escherichia coli, etc. It can be divided into clinical and subclinical types (hidden mastitis). Clinical mastitis usually has obvious inflammatory symptoms, such as redness, swelling, warmth and pain in the breast, milk degeneration, possible curds or flocs, reduced or stopped lactation, which will lead to a significant decrease in milk production. Severe cases will also cause no milk or loss of udder function. Timely treatment is required and the milk produced during treatment cannot be eaten and must be discarded. In dairy farming and production, the cost of treatment will increase at the least, and the use of antibiotics for treatment may have drug residue problems. In severe cases, it will lead to the loss of lactation function of dairy cows and affect their production life. Therefore, breeding to improve mastitis resistance has become an important goal of balanced breeding for dairy cows.

[0003] Molecular breeding has gradually become a trend in biological breeding, among which breeding using single nucleotide polymorphism (SNP) information has the advantages of easy detection and genetic stability. Using SNP for disease-resistant breeding of dairy cows meets the needs of the times, can further meet the strategic needs of the national seed industry revitalization, can reduce the use of veterinary drugs to achieve green agricultural development, and has important strategic significance and application value.

[0004] The resistance genes currently reported to be associated with mastitis in dairy cows include BRCA1 Genes, etc., but no reports have been made on the relationship between dairy cow mastitis and genes HK1 , ATG5 , POLR3C , RSAD2 , LRIG2 relationship. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a gene, a molecular marker and an application related to cow mastitis resistance.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: Provide a gene related to cow mastitis resistance, including: gene HK1 , POLR3C , RSAD2 , LRIG2 ,Gene HK1 The sequence number is GCF_002263795.3, gene POLR3C The sequence number is NC_091688, gene RSAD2The sequence number is NC_091698, gene LRIG2 The serial number is NC_091688.1.

[0007] Primers for amplifying the above genes related to cow mastitis resistance, for amplifying genes HK1 The primer sequences are: HK1 -F: 5'-CTACCTGTTCCCGGAGGGTA-3' (SEQ ID NO.1); HK1 -R: 5'-TCCATTCCATTTGAGCGCCT-3' (SEQ ID NO. 2); For gene amplification POLR3C The primer sequences are: POLR3C -F: 5'-GGAATGGGTCCTCAGCTTCC-3' (SEQ ID NO.3); POLR3C -R: 5'-AGACAACCGAACAGCGACAA-3' (SEQ ID NO.4); For gene amplification RSAD2 The primer sequences are: RSAD2 -F: 5'-TACAGTGCCATCCCAGCAAG-3' (SEQ ID NO.5); RSAD2-R :5'-TTTTTCCTTCCTCCCAGGCAT-3' (SEQ ID NO.6); For gene amplification LRIG2 The primer sequences are: LRIG2 -F: 5'-GGAATGGGTCCTCAGCTTCC-3' (SEQ ID NO.7); LRIG2 -F: 5'-AGACAACCGAACAGCGACAA-3' (SEQ ID NO. 8).

[0008] A kit comprises primers of the above-mentioned gene related to mastitis resistance in dairy cows.

[0009] Molecular marker associated with resistance to mastitis in dairy cows, the molecular marker being the above gene HK1 When the polymorphism is G / A at position 25767925 and the polymorphic site is A, it indicates mastitis resistance; or The above genes POLR3CAt position 21514654, the polymorphism is C / T, and when the polymorphic site is T, it is mastitis resistance; or The above genes RSAD2 At position 90032843, the polymorphism is C / A, and when the polymorphic site is A, it is mastitis resistance; or The above genes LRIG2 At position 30210467, the polymorphism is C / T. When the polymorphic site is T, it is mastitis resistance.

[0010] Application of the reagent for detecting the gene related to cow mastitis resistance or the reagent for molecular marker in the preparation of a reagent for detecting cow mastitis resistance.

[0011] Application of the above genes or molecular markers related to mastitis resistance in dairy cows in genetic breeding of mastitis-resistant dairy cows.

[0012] The present invention has the following beneficial effects: The present invention uses the genome sequence of dairy cows and whole genome resequencing to find the selection signals and genetic variations of significant allele frequency differences between the populations of resistant and susceptible dairy cows. The genes related to dairy cow mastitis resistance were screened out through selection signal analysis, namely, genes HK1 , ATG5 , POLR3C , RSAD2 , LRIG2 , and identified the SNP sites of these genes. These SNP molecular markers can accurately predict the mastitis resistance characteristics of dairy cows under different genetic backgrounds, and can be used to identify dairy cows with high mastitis resistance traits and improve the efficiency of molecular design breeding. And according to the identification results of the above SNP sites, breeders can select favorable genotype individuals for breeding, thereby improving the mastitis resistance traits of dairy cow groups, improving the mastitis resistance of dairy cow groups, reducing treatment costs, and improving the economic benefits of pastures. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Between Group 1 and Group 2 susceptible cattle F Select Manhattan plot for signal analysis.

[0014] Figure 2 The difference between group 2 resistant cattle and group 2 susceptible cattle F Select Manhattan plot for signal analysis.

[0015] Figure 3 Group 2 disease-resistant cattle and Group 2 susceptible cattle HK1 Genetic F value.

[0016] Figure 4 Group 2 susceptible cattle and Group 1 resistant cattle HK1 Genetic F value.

[0017] Figure 5 Group 2 disease-resistant cattle and Group 2 susceptible cattle POLR3C Genetic F value.

[0018] Figure 6 Group 2 susceptible cattle and Group 1 resistant cattle POLR3C Genetic F value.

[0019] Figure 7 Group 2 susceptible cattle and Group 1 resistant cattle RSAD2 Genetic F value.

[0020] Figure 8 Group 2 susceptible cattle and Group 1 resistant cattle LRIG2 Genetic F value. DETAILED DESCRIPTION

[0021] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] Embodiment 1: 1. Materials and Methods 1. Experimental Animals The present invention selects 69 dairy cows as research objects, wherein frozen semen samples of 9 anti-mastitis cows are used as group 1, and whole blood samples of 60 cows are used as group 2, wherein the 60 cows include 30 anti-mastitis cows and 30 mastitis susceptible cows. The screening of disease resistance and susceptibility is based on the same trait of three generations.

[0023] 2. Test methods (1) Extraction of DNA from frozen bovine semen The phenol-imitation extraction method is used to extract the DNA from frozen bovine semen. The specific process is as follows: ① Take 100-125μL frozen semen, add 400μL physiological saline, mix thoroughly by inversion, centrifuge at 12000rpm for 10min, discard the supernatant, repeat this step once to obtain the precipitate.

[0024] ② Add 600 μL frozen sperm DNA extract and 10 μL proteinase K to the precipitate, shake and mix, and digest at 56°C overnight.

[0025] ③ Add 500 μL of a mixed solution of phenol: chloroform: isoamyl alcohol (25:24:1), invert to mix, and centrifuge at 12,000 rpm for 10 min.

[0026] ④ Take the supernatant, add 500 μL of a mixed solution of chloroform:isoamyl alcohol (24:1), invert to mix, and centrifuge at 12000 rpm for 10 min.

[0027] ⑤ Take the supernatant, add about 800 μL of ice-cold anhydrous ethanol, invert and mix, then white flocculent precipitate can be seen, and finally centrifuge at 4℃ 12000rpm for 2min.

[0028] ⑥ Rinse with 800 μL 70% ethanol, centrifuge at 12000 rpm at 4°C for 2 min, and repeat this step once.

[0029] ⑦ Dry at room temperature and dissolve in water to 50-100μL.

[0030] (2) Extraction of blood genomic DNA Blood genomic DNA was extracted using Tiangen Blood Genomic DNA Extraction Kit (DP348).

[0031] (3) Whole genome resequencing The extracted DNA samples were subjected to 1% agarose gel electrophoresis to detect the DNA quality, and the concentration of the extracted genomic DNA was tested using a micro-nucleic acid concentration meter. After the DNA bands were intact and non-degraded and the concentration was >50 ng / μL, Beijing Berry Genomics Biotechnology Co., Ltd. randomly sheared the genomic DNA that passed the quality inspection, recovered the required length by electrophoresis, added adapters, and prepared the library. Each library that passed the quality inspection was subjected to whole-genome resequencing with a data volume of 30 GB (about 30×) using the Illumina X plus platform PE150 sequencing method.

[0032] (4) Sequencing data quality control Trimmomatic v0.39 software was used to remove the connector files of the raw sequencing data and filter out reads with low quality values. BWA 0.7.17-r1188 was used to align with the reference genome (ASM130575 v1). The number of reads, alignment ratio, and sequencing depth were completed by samtools v1.10. GATK v4.1.4.1 was used to sort, deduplicate, and detect variants in the alignment files. Haplotype Caller was used to detect SNPs in each sample, and then the Combine GVCFs tool was used to merge the variants of all samples. Genotype GVCFs was used to detect variants in all samples. After the detection, the parameters: QD < 2.0 || MQ < 40.0 || FS > 60.0 || SOR > 3.0 || MQRank Sum < -12.5 || ReadPosRank Sum < -8.0 were used to filter out variant sites with low quality values. PLINKv1.9 was used to perform quality control of variants, including samples with SNP detection rate lower than 80% (--mind0.2), SNPs with more than 10% missing data (--geno0.1), SNPs with MAF lower than 5% (--MAF0.05), and Hardy-Weinberg test value less than 1e -07 (--hwe0.0000001) SNPs were removed.

[0033] (5) Select signal analysis A population differentiation-based approach was used F (Wright's fixation index) was used to analyze the selection signal of two groups of susceptible cattle in group 1 and group 2, and resistant cattle in group 2 and susceptible cattle in group 2. VCFtools

[129] was used to calculate F The parameters were -fst-window-size 50000 --fst-window-step 10000. The selection signal analysis results were then plotted using the Cario and qqman packages in R studio to draw a Manhattan plot. The top 1% of the selected signals were annotated using bedtools v2.29.2, and the DAVID (https: / / david.ncifcrf.gov / ) website was used to perform gene enrichment analysis and screen candidate genes associated with anti-mastitis traits.

[0034] 2. Results 1. Whole genome resequencing results The quality control results of whole genome sequencing of 69 dairy cows showed that after removing adapter contamination, removing low-quality bases, and removing reads containing N ratio greater than 5%, the alignment rate between the raw data of all samples and the reference genome exceeded 99%, and more than 90% of the base quality values ​​of the samples after filtering and quality control reached Q30 accuracy, indicating that the quality of genome sequencing was good.

[0035] 2. Select signal analysis results Between Group 1 and Group 2 susceptible cattle F Select the Manhattan plot for signal analysis such as Figure 1 As shown. Figure 1 It can be seen that there are 1041 genes in the top 1% of selection signal regions.

[0036] Between group 2 resistant cattle and group 2 susceptible cattle F Select the Manhattan plot for signal analysis such as Figure 2 As shown. Figure 2 It can be seen that there are 1534 genes in the top 1% of selection signal regions.

[0037] 3. Screening of candidate genes Based on the population differentiation index F , can detect the difference in allele frequency between groups, effectively detect the selected sites in the group, and then obtain the relevant SNP information after detecting the selected sites in the test group, and then use bedtools v2.29.2 for gene annotation to obtain candidate genes. The specific candidate genes are as follows: ① HK1 (hexokinase1), chr28: 25677522-25768161 Gene HK1 At position 25767925, the polymorphism is G / A. When the polymorphic site is A, it is mastitis resistance.

[0038] Group 2 (resistant) × Group 2 (susceptible): highest peak within the gene F =0.15, see Figure 3 .

[0039] Group 2 (susceptible) × Group 1 (resistant): highest peak within the gene F =0.28, see Figure 4 .

[0040] HK1 Hexokinase 1 catalyzes the phosphorylation of various hexoses to hexose 6-phosphates. Involved in innate immunity and inflammation by acting as a pattern recognition receptor for bacterial peptidoglycan. When the N-acetyl-D-glucosamine component of bacterial peptidoglycan is released into the cytoplasm, it inhibits the hexokinase activity of HK1 and causes its dissociation from the mitochondrial outer membrane, thereby activating the NLRP3 inflammasome (By similarity). Phenotypic traits are associated with the immune system, and this gene is involved in the positive regulation of cytokine production in immune responses.

[0041] ② POLR3C ,chr3:21519780-21532978 Gene POLR3C At position 21514654, the polymorphism is C / T. When the polymorphic site is T, it indicates mastitis resistance.

[0042] Group 2 (resistant) × Group 2 (susceptible): highest peak within the gene F =0.25, see Figure 5 .

[0043] Group 2 (susceptible) × Group 1 (resistant): highest peak within the gene F =0.15, see Figure 6 .

[0044] This gene is part of the RNA polymerase III complex and is involved in the positive regulation of the innate immune response and the production of interferon-β.

[0045] ③ RSAD2 ,chr11:90038994-90056291 Gene RSAD2 At position 90032843, the polymorphism is C / A, and the polymorphic site is A, which is mastitis resistance.

[0046] Group 2 (resistant) × Group 2 (susceptible): highest peak within the gene F =0.18.

[0047] Group 2 (susceptible) × Group 1 (resistant): highest peak within the gene F =0.24, see Figure 7 .

[0048] RSAD2 (a free radical containing 2 S-adenosylmethionine domains) is an interferon-induced antiviral protein encoding gene. Its related pathways include cytokine signaling in the immune system and the antiviral mechanism of IFN-stimulated genes. It promotes T cell receptor (TCR)-mediated GATA3 activation and optimal T helper cell 2 (Th2) cytokine production by regulating NFKB1 and JUNB activity, and can inhibit the secretion of soluble proteins.

[0049] ④ LRIG2 ,chr3:30209449-30272471 Gene LRIG2 At position 30210467, the polymorphism is C / T. When the polymorphic site is T, it is mastitis resistance.

[0050] Group 2 (susceptible) × Group 1 (resistant): highest peak within the gene F =0.33, see Figure 8 .

[0051] This gene encodes a transmembrane protein containing a leucine-rich repeat sequence and an immunoglobulin-like domain. The encoded protein promotes epidermal growth factor signaling, leading to increased proliferation, and its phenotypic traits are related to the immune system.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A gene associated with resistance to mastitis in dairy cows, characterized in that: include: Gene HK1 , POLR3C , RSAD2 , LRIG2 ,Gene HK1 The sequence number is GCF_002263795.3, gene POLR3C The sequence number is NC_091688, gene RSAD2 The sequence number is NC_091698, gene LRIG2 The serial number is NC_091688.

1.

2. The primers for amplifying the gene associated with cow mastitis resistance according to claim 1, characterized in that: For gene amplification HK1 The primer sequences are: HK1 -F:5’-CTACCTGTTCCCGGAGGGTA-3’; HK1 -R:5’-TCCATTCCATTTGAGCGCCT-3’; For gene amplification POLR3C The primer sequences are: POLR3C -F:5'-GGAATGGGTCCTCAGCTTCC-3': POLR3C -R:5'-AGACAACCGAACAGCGACAA-3'; For gene amplification RSAD2 The primer sequences are: RSAD2 -F:5’-TACAGTGCCATCCCAGCAAG-3’; RSAD2-R :5’-TTTTTCCTCTCTCCCAGGCAT-3’; For gene amplification LRIG2 The primer sequences are: LRIG2 -F:5'-GGAATGGGTCCTCAGCTTCC-3'; LRIG2 -F: 5'-AGACAACCGAACAGCGACAA-3'.

3. A kit, characterized in that: The invention relates to a primer comprising the gene related to the resistance to mastitis in dairy cows as claimed in claim 2.

4. A molecular marker associated with resistance to mastitis in dairy cows, characterized in that: The molecular marker is the gene described in claim 1 HK1 At position 25767925, the polymorphism is G / A; or The gene of claim 1 POLR3C The polymorphism at position 21514654 is C / T; or The gene of claim 1 RSAD2 At position 90032843, the polymorphism is C / A; or The gene of claim 1 LRIG2 At position 30210467, the polymorphism is C / T.

5. The molecular marker associated with cow mastitis resistance according to claim 4, characterized in that: Gene HK1 When the polymorphic site is A, it is mastitis resistance; POLR3C When the polymorphic site is T, it is mastitis resistance; RSAD2 When the polymorphic site is A, it is mastitis resistance; LRIG2 When the polymorphic site is T, it indicates mastitis resistance.

6. Use of a reagent for detecting a gene associated with cow mastitis resistance according to claim 1 or a reagent for detecting a molecular marker according to claim 4 or 5 in the preparation of a reagent for detecting cow mastitis resistance.

7. Use of the gene associated with cow mastitis resistance according to claim 1 or the molecular marker according to claim 4 or 5 in genetic breeding of mastitis-resistant cows.

Images