Molecular marker related to number of blood platelets and somatic cell score of dairy cow and application of molecular marker

Abstract

The invention belongs to the technical field of molecular breeding, and particularly relates to a molecular marker related to the number of blood platelets and somatic cell scores of dairy cows and application of the molecular marker. Whole genome re-sequencing and genotype analysis are carried out on 348 Holstein dairy cows, and GWAS and haplotype correlation analysis is combined, so that a haplotype formed by two SNP sites on an NKAIN2 gene at 27230850 bp and 27231227 bp of a ninth chromosome of the dairy cows is found to be remarkably related to the number of platelets and somatic cell scores. The platelet number estimation breeding value of a haplotype GA individual is remarkably lower than that of an AG individual, and the somatic cell score estimation breeding value is remarkably lower than that of the AG individual. The molecular marker provided by the invention can be used for auxiliary screening of dairy cows with relatively low somatic cell scores and suitable platelet levels, improves the disease resistance and production benefits of the dairy cows, and is suitable for molecular marker-assisted breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular breeding, and relates to a molecular marker related to the number of platelets and somatic cell score of a dairy cow and application thereof. BACKGROUND

[0002] Dairy cow is one of the core breeds in modern animal husbandry in China, and cow milk is rich in nutritional value and plays an important role in human diet. At present, dairy farming has become an important pillar of agricultural economy. However, long-term high-intensity selection of economic traits has led to a general decline in the resistance of dairy cows to diseases, among which respiratory diseases, digestive system diseases and reproductive system diseases have become the main reasons affecting the lactation performance of dairy cows or directly leading to the death of dairy cows, causing great economic losses to dairy farming. Therefore, genetic selection of disease resistance of dairy cows is an important way to improve the production efficiency of dairy cows in the future.

[0003] Blood cells play a key role in the immune response and disease resistance of animals, mainly including three main cell types of white blood cells, red blood cells and platelets. Among them, platelets are nuclear cell fragments produced by megakaryocytes, and mainly participate in the pathophysiological processes of coagulation, wound healing and thrombosis. Studies have shown that platelets and their activated products play an important role in inflammatory reactions. Activated platelets form platelet-leukocyte aggregates, adhere to inflammatory cells and secrete pro-inflammatory mediators stored in platelets, thereby regulating inflammation. Therefore, platelet count is usually increased in mild or early inflammatory reactions, but it is reduced in severe systemic inflammation due to excessive consumption or bone marrow suppression. In general, the normal value range of platelets of healthy Holstein dairy cows is 273.15-980.70 (10 9 / L), and both increase and decrease outside the normal value range will affect the health of dairy cows. In the field of agricultural breeding, abnormal reduction of platelets is an important indication of various infectious diseases, which are commonly seen in salmonellosis, leptospirosis, babesiosis, theileriosis and bovine viral diarrhea virus infection; platelet reduction accompanied by leukopenia and bone marrow suppression is also an important indication for the clinical diagnosis of anemia in newborn calves. In addition, cold and heat stress can also significantly affect the platelet count of dairy cows. In the field of human medicine, changes in platelet levels have been confirmed to be closely related to inflammatory bowel disease, pulmonary inflammatory disease and acute mastitis during lactation. When acute mastitis occurs, the release of inflammatory mediators increases due to platelet activation, and the inflammatory response is enhanced, so anti-platelet therapy has become an important direction for subsequent reduction of inflammatory response in patients with acute mastitis. Although there is no research report on the treatment of inflammatory diseases in dairy cows, it may be an important target for the treatment of various inflammatory diseases, thereby blocking the vicious cycle caused by the development of inflammatory response.

[0004] The difficulty of the anti-disease breeding technology lies in the accurate positioning of the quantitative trait loci (QTL). At present, the method for positioning the QTL of the dairy cow mainly relies on genome-wide association studies (GWAS), and the method has become the mainstream means for genetic analysis of important economic traits of the dairy cow. The GWAS method can identify SNP markers of known genes and screen SNP markers of unknown genes, and the limitation of the GWAS method is that most of the SNP markers identified by the GWAS method are located in the intergenic region, or the genetic effect is small, the repeatability is low, and it is difficult to be further used for breeding application. Under the background, the application will use the gene level association analysis, linkage disequilibrium analysis and haplotype association analysis and other technical means to accurately position the target traits according to the GWAS results. Through the screening method, the QTL with higher accuracy and reliability can be obtained, which can effectively alleviate the shortage of the QTL of the disease resistance traits of the dairy cow, and gradually meet the needs of the molecular breeding of the dairy cow. SUMMARY

[0005] The application aims to provide a molecular marker related to the number of platelets and somatic cell score of a dairy cow and application thereof. The application identifies a QTL affecting the number of platelets of the dairy cow through genome-wide association analysis and haplotype analysis, and finds that the QTL is related to the somatic cell score of the dairy cow through association analysis with the lactation traits, and identifies two SNP sites (g.27230850 G>A and g.27231227 A>G) of the dairy cow in the QTL region, which constitute a haplotype block, wherein the GA type haplotype is significantly related to the lower number of platelets and somatic cell score. The molecular marker can be used for assisting breeding of the dairy cow with strong disease resistance. NKAIN2 The application aims to provide a molecular marker related to the number of platelets and somatic cell score of a dairy cow and application thereof. The application identifies a QTL affecting the number of platelets of the dairy cow through genome-wide association analysis and haplotype analysis, and finds that the QTL is related to the somatic cell score of the dairy cow through association analysis with the lactation traits, and identifies two SNP sites (g.27230850 G>A and g.27231227 A>G) of the dairy cow in the QTL region, which constitute a haplotype block, wherein the GA type haplotype is significantly related to the lower number of platelets and somatic cell score. The molecular marker can be used for assisting breeding of the dairy cow with strong disease resistance.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is: The application provides a molecular marker for identifying or assisting in identifying the number of platelets or the somatic cell score of a dairy cow, wherein the molecular marker is located at the SNP site of the dairy cow genome ARS-UCD2.0 version chromosome 9 NKAIN2 Gene region, respectively: g.27230850 G>A and g.27231227 A>G; the nucleotide sequence of chromosome 9 27230850 bp to 27231227 bp is SEQ ID NO. 1.

[0007] It is further explained that the two SNP sites constitute a haplotype block, and contain two haplotypes of GA type and AG type.

[0008] The application also provides application of a substance for detecting polymorphism or genotype of the SNP site in identifying or assisting in identifying platelet number or somatic cell score of a dairy cow.

[0009] The application also provides application of a substance for detecting the haplotype in identifying or assisting in identifying platelet number or somatic cell score of a dairy cow.

[0010] It is further illustrated that the haplotype GA type is related to lower estimated breeding value of platelet number and lower estimated breeding value of somatic cell score.

[0011] It is further illustrated that the detection substance includes primers, probes, chips or sequencing reagents for amplifying a genomic fragment containing the SNP site.

[0012] The application also provides a kit for dairy cow breeding, comprising a substance for detecting the SNP site or haplotype.

[0013] It is further illustrated that the kit is used for screening dairy cows with lower somatic cell score and suitable platelet level.

[0014] The application also provides a method for identifying or assisting in identifying platelet number or somatic cell score of a dairy cow, comprising detecting genotype or haplotype of the SNP site in a dairy cow to be tested, and judging the breeding value of platelet number or somatic cell score of the dairy cow according to the genotype or haplotype.

[0015] It is further illustrated that the method is used for disease-resistant breeding of a dairy cow, and individuals with GA haplotype are retained to improve disease resistance of the dairy cow.

[0016] By adopting the technical scheme, the application has the following beneficial effects: The application finds a QTL affecting platelet number and somatic cell score of a dairy cow through genome-wide association analysis (GWAS) and haplotype analysis. NKAIN2 The QTL is two sites at 27230850 bp and 27231227 bp of chromosome 9 of ARS-UCD version 2.0 genome of a dairy cow, is located on an intron of a gene, is closely linked, and contains two haplotypes of GA type and AG type. P The estimated breeding value of platelet number of a Holstein dairy cow with GA haplotype is significantly lower than that of a dairy cow individual with AG haplotype ( P <0.01), and the estimated breeding value of somatic cell score is significantly lower than that of a dairy cow individual with AG haplotype ( P < 0.05). The QTL can be used for disease-resistant breeding of a dairy cow, and individuals with GA haplotype are retained to improve disease resistance of the dairy cow, thereby reducing economic loss of a farm. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1This is a GWAS Manhattan plot of the quantitative trait of platelets in dairy cows.

[0018] Figure 2 yes NKAIN2 Distribution map of gene linkage disequilibrium regions.

[0019] Figure 3 This is a comparison chart of estimated breeding values ​​of platelets in individuals with two haplotypes of block43.

[0020] This indicates a highly significant difference between the two genotypes. Detailed Implementation

[0021] The specific implementation of the invention will be further described below with reference to the accompanying drawings. Example 1

[0022] (a) Experimental Design: 1. Sample collection and DNA preparation: Blood samples were collected from 348 Holstein dairy cows via tail vein, stored in EDTA anticoagulant tubes at 4°C, and total DNA was extracted using the Tiangen Blood Genomic DNA Extraction Kit.

[0023] 2. Phenotypic Collection and Breeding Value Estimation: Lactation performance data (including 305-day milk yield, milk fat percentage, milk protein percentage, and somatic cell count) of 348 Holstein dairy cows were compiled, and individual blood five-part differential assays were performed using the ADVIA® 2120i blood analyzer. Estimated breeding values ​​(EBV) for blood five-part differential and lactation traits were calculated using a single-trait animal model. The model is as follows: y = Xb + Zg + e , in y These are blood phenotypic values. X For the fixed effects matrix, b Fixed-effect variables include batch number (two levels, including first and second batches), parity (four levels, including first, second, third and fourth or more parities), and lactation stage (four levels, including dry period, early lactation, mid-lactation and late lactation). Z The random effects matrix, g It is an additive effect on individuals; e This is due to the residual effect.

[0024] 3. SNP genotyping: A 10X depth whole-genome resequencing strategy was used to sequence 348 individuals. SNP genotyping was performed using GATK 4.2 software, followed by genotyping using Beagle 5.4 software. Population SNP filtering was performed using PLINK software. The filtering criteria were as follows: individual call rate of ≥97%; SNP detection rate of ≥95%; minimum allele frequency (MAF) greater than 0.05; Hardy-Weinberg equilibrium test. P Value less than 10 −6 After filtering, 1,030,098 SNPs were obtained and can be used for subsequent analysis.

[0025] 4. Population structure and linkage disequilibrium analysis: Principal component analysis (PCA) was performed on the population using PLINK software to evaluate the population's genetic structure; PopLDdecay software was used to evaluate the degree of genome-wide linkage disequilibrium (LD) in the population, and to assess the efficiency and accuracy of the association analysis.

[0026] 5. Genome-wide association analysis (SNP-GWAS) at the SNP level: GWAS analysis of each blood trait was performed using the FarmCPU method in rMVP software. The kinship matrix and four principal components were used as covariates. Boferroni was employed to analyze the SNP-GWAS. P The value is corrected. The threshold for the GWAS signal is selected as 0.05 / N , N It equals 1030098.

[0027] 6. Genome-based GWAS at the gene level: Extracting the location information of each SNP locus from step (V) of the SNP-GWAS results and P Based on SNP locus location information and the bovine ARS-UCD 2.0 whole-genome annotation file, SNPs were annotated, resulting in 21,521 candidate associated genes. Gene-level association analysis was performed using the MAGMA software gene-based analysis model, and after model filtering, 14,674 genes meeting the analysis requirements were obtained. Boferroni was used to analyze... P The value is corrected. The threshold for the GWAS signal is selected as 0.05 / N , N It equals 14674.

[0028] 7. Haplotype Association Analysis: Based on the gene-based GWAS results, linkage disequilibrium regions within genes were constructed using LD BlockShow software. Haplotypes of each linkage disequilibrium region were constructed using the geneHapR software package, and association analysis was performed with estimated breeding values ​​for blood and lactation traits. Haplotypes associated with phenotypic traits ( P Sites containing <0.05 were identified as candidate QTLs.

[0029] (II) Test Results: 1. Platelet count-related gene-based GWAS signal screening Manhattan plot, representing the effect value of genetic markers, i.e., the whole genome as determined by the F-test. P Values ​​sorted by physical location on chromosomes, with the horizontal axis representing the physical location on the chromosome of the genome and the vertical axis representing -log. 10 P , P The smaller the value, the stronger the correlation, which is reflected in the larger the ordinate. In the Manhattan plot, the horizontal dashed line represents the significance level; when -log 10 P When the SNP value is greater than 5, it is considered to be significantly associated with the trait.

[0030] Gene-based GWAS results for estimating breeding values ​​based on platelet count are as follows: Figure 1 As shown, three genes are significantly associated with platelet count in dairy cows, located on chromosomes 4, 9, and 11, respectively. NKAIN2 The gene is located on chromosome 9 at positions 26362416-27543450 bp, containing a total of 721 SNP sites.

[0031] 2. NKAIN2 Haplotype construction of genes and their association with platelets Haplotype identification results as follows Figure 2 As shown, NKAIN2 The gene contains 52 linked regions, the largest of which is located at 27449730-27521116 bp on chromosome 9, containing 225 loci.

[0032] Haplotypes were constructed for each linkage region and correlated with estimated platelet counts in breeding studies. The results showed that the haplotype (block 43, see SEQ ID NO. 1) formed by two sites at 27230850 bp and 27231227 bp was highly significantly associated with the estimated platelet counts in breeding studies. Figure 3The mean estimated breeding value of platelet count for GA haplotype individuals in this block was -42.08, while the mean estimated breeding value of platelet count for AG haplotype individuals was 487.97. There was a highly significant difference in platelet count between the two haplotypes. P < 0.0001), indicating that under the same feeding conditions and physiological status, the platelet count of GA individuals (average 350.40 × 10⁻⁶) is significantly lower. 9 The mean value ( / L) was significantly lower than that of AG individuals (mean value was 528.25 × 10⁻⁶). 9 / L).

[0033] 3. NKAIN2 The Influence of Two Haplotypes of Gene Block 43 on Estimated Breeding Values ​​of Dairy Cow Lactation Performance Further analysis of the effects of the two haplotypes on dairy cow lactation performance in block 43 was conducted, and the results are shown in Table 1. Among the four traits of milk yield, milk fat percentage, milk protein percentage, and somatic cell score at 305 days, the estimated breeding value of the cell score for haplotype GA(H001) was significantly lower than that for haplotype AG(H002). P < 0.05), while other traits showed no significant differences. This result indicates that, under the same feeding conditions and physiological status, the somatic cell score of GA(H001) individuals (mean 2.9209) was significantly lower than that of AG(H002) individuals (mean 2.9287).

[0034]

[0035] Note: MY305 is milk production in 305 days, FP is milk fat percentage, PP is milk protein percentage, SCS is somatic cell score, and all phenotypes are expressed as "least square mean ± standard error". Different superscripts in the same column indicate significant differences.

[0036] In summary, GA haplotype individuals are significantly superior to AG haplotype individuals in both platelet count and somatic cell score, and can be used for disease-resistant breeding. Example 2

[0037] A method for assisting in the early selection of highly disease-resistant dairy cows involves detecting two loci at 27230850 bp and 27231227 bp on chromosome 9 of the dairy cow genome (ARS-UCD 2.0 version), located at... NKAIN2 On the intron of the gene, these two loci form a linked block containing two haplotypes: GA and AG. Individuals carrying the GA haplotype are selected as parents for further selection. Example 3

[0038] Applications of molecular markers: The specific primer pair is used for PCR amplification and sequencing of the fragment containing g.27230850 G>A and g.27231227 A>G sites, so that the haplotype can be accurately typed, the individual with AG haplotype is eliminated, the individual with GA haplotype is reserved, and early selection of high disease-resistant dairy cows is assisted. Example 4

[0039] Preparation of the kit: The primer, Taq enzyme, dNTPs, buffer and the like in example 3 are combined to form a PCR kit, which can be used for rapid detection of the haplotype of the two sites g.27230850 G>A and g.27231227 A>G of dairy cows, elimination of the individual with AG haplotype, reservation of the individual with GA haplotype, and assistance in disease-resistant breeding.

[0040] The above description is a detailed description of the preferred embodiments of the present application, but the embodiments are not used to limit the scope of the patent application of the present application. Any equivalent changes or modifications made under the technical spirit of the present application should belong to the patent scope covered by the present application.

Claims

1. A molecular marker for identifying or assisting in identifying the number of platelets or the somatic cell score of a dairy cow, characterized in that, The molecular marker is located on chromosome 9 of the genome of the cow ARS-UCD version 2.0 NKAIN2 SNP sites of the gene region are g.27230850 G>A and g.27231227 A>G respectively; the nucleotide sequence of chromosome 9 of the cow from 27230850 bp to 27231227 bp is SEQ ID NO.

1.

2. The molecular marker of claim 1, wherein The two SNP sites constitute a haplotype block, including two haplotypes of GA type and AG type.

3. Use of a substance for detecting polymorphism or genotype of the SNP site of claim 1 in identifying or assisting in identifying the platelet number or somatic cell score of a dairy cow.

4. Use of a substance for detecting the haplotype of claim 2 in identifying or assisting in identifying the platelet number or somatic cell score of a dairy cow.

5. Use according to claim 3 or 4, characterized in that, The haplotype GA type is associated with lower estimated breeding value of platelet number and lower estimated breeding value of somatic cell score.

6. Use according to claim 3 or 4, wherein the compound is ###0002### The detection substance includes primers, probes, chips or sequencing reagents for amplifying the genomic fragment containing the SNP site.

7. A kit for dairy cattle breeding, characterized in that, A substance containing the SNP site or haplotype of claim 1 or 2 for detection.

8. The kit of claim 7, wherein For screening dairy cows with lower somatic cell score and suitable platelet level.

9. A method of identifying or aiding in the identification of the number of platelets or the somatic cell score of a dairy cow, characterized in that, Including detecting the genotype or haplotype of the SNP site of claim 1 or 2 in the dairy cow to be tested, and judging its platelet number or somatic cell score according to its genotype or haplotype.

10. Use of the method of claim 9 in the breeding of disease-resistant dairy cows, retaining individuals with GA haplotype to improve the disease resistance of dairy cows.

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