WO2021248793A1 - Method for screening ketosis-resistant molecular markers of dairy cows and application thereof - Google Patents

Abstract

Provided are a method for screening ketosis-resistant molecular markers of dairy cows and an application thereof, which belong to the technical field of molecular genetic biology. Taking healthy and ketosis dairy cows as samples, chip sequencing, data filling, selection signal analysis, and whole-genome association analysis, and the like are performed to screen out a key gene APOA1 that affects the resistance of Chinese Holstein cattle to ketosis. The single nucleotide polymorphism site g.-572A>G is obtained by screening in the APOA1 gene promoter region. By means of association analysis, promoter activity analysis and other methods, it is obtained that the concentration of β-hydroxybutyric acid in the blood of GG genotype dairy cows is significantly lower than that of the AA genotype, and the promoter activity of GG genotype individuals is significantly higher than that of AA genotype individuals. By using the technical solution, genotyping the APOA1 gene promoter region site g.-572A>G may screen out dairy cow individuals that have strong ketosis resistance, thus achieving the purpose of saving on breeding costs.

Description

Method for screening dairy cow ketosis resistance molecular markers and application thereof Technical field

The invention belongs to the technical field of molecular genetic biology, and specifically relates to a method for screening a molecular marker for ketosis resistance in dairy cows and an application thereof.

Background technique

Disclosure of the background information is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art known to those of ordinary skill in the art.

In recent decades, the degree of intensive feeding in the dairy industry has continued to increase, and the pursuit of high yields has led to increasingly prominent postpartum diseases in dairy cows. Dairy cow ketosis is a metabolic disease caused by insufficient supply of carbohydrates such as sugars and volatile fatty acids to cause dysfunction of postpartum dairy cows. It mostly occurs in high-yielding dairy cows with good lactation performance (Hibbitt 1979). Ketosis is one of the most common and expensive metabolic diseases in dairy farms, with a prevalence of approximately 30%-40% (Zhang and Ametaj, 2017). Cows suffering from ketosis have reduced milk production, reproductive performance and immunity, and are at higher risk of other perinatal diseases (McArt et al., 2013). Duffield pointed out that the combined losses of a case of ketosis cattle ranged from 50 to 100 US dollars (Duffield, 2000). Ketosis is characterized by increased levels of ketone bodies in milk, urine, and blood of cows. Clinically, acetoacetate, β-hydroxybutyric acid (BHBA) and acetone are collectively referred to as ketone bodies. Beta-hydroxybutyric acid is the most common ketone body for diagnosing ketosis in dairy cows (Oetzel, 2004). Plasma β-hydroxybutyric acid concentration ≥1.2mmol/L is the gold standard for judging whether cows are suffering from ketosis (McArt et al., 2011).

Dairy cows are resistant to ketosis, and there are large differences between individuals. The inventor found that the heritability of ketosis resistance is low, about 0.02-0.16. It is extremely difficult to rely on phenotypic conventional breeding methods for selection, and molecular breeding strategies have more advantages for traits with low heritability and can be very good. Solve this problem (Koeck et al., 2012; Koeck et al., 2014). Foreign researchers have also regarded ketosis resistance as a hot trait in dairy cattle breeding and have begun to pay attention (Kroezen et al., 2018; Parker Gaddis et al., 2018). Therefore, further mining the functional genes and markers that affect the formation of ketosis resistance in dairy cows and clarifying their molecular regulation mechanisms are of great significance and value for the molecular breeding of high ketosis resistant cattle.

Summary of the invention

In view of the above-mentioned prior art, the present invention provides a method and application for screening bovine ketosis resistance molecular markers. The present invention aims at the genetic characteristics of Chinese Holstein dairy cows’ ketosis, from the perspective of genome evolution and selection and disease susceptibility. Consider using the cattle 150K gene chip to sequence the blood samples of ketosis and healthy Chinese Holstein dairy cows, and integrate the Fst, XPEHH, and XPCLR three genomic selection signal analysis methods as well as the GEMMA and PLINK whole-genome association analysis methods, and successfully screened dairy cows The ketosis resistance gene and its molecular markers, and the establishment of corresponding detection methods, have good practical application value.

In order to achieve the above objectives, the present invention relates to the following technical solutions:

The first aspect of the present invention provides a method for screening a molecular marker for ketosis resistance in dairy cows, the method at least comprising:

Analyze DNA samples of healthy and ketotic cows based on SNP chip detection;

Analysis based on Fst, XPEHH and XPCLR genome selection signal analysis methods and GEMMA and PLINK genome-wide association analysis methods; screening SNPs and candidate genes that are significantly positively selected and significantly associated with ketosis, and integrate gene function annotations Screen SNP molecular markers for ketosis resistance.

Further, based on the above method, the present invention screened and identified the APOA1 gene as a key gene for ketosis resistance in dairy cows, and therefore can be used as a molecular marker for ketosis resistance in dairy cows; furthermore, molecular markers for ketosis resistance in dairy cows also include A single nucleotide polymorphism in the gene: g.-572A>G. Experiments have shown that the concentration of β-hydroxybutyric acid in the blood of GG type cows with APOA1 gene is significantly lower than that of AA genotype. And the luciferase activity test showed that the promoter activity of APOA1 gene GG type was significantly higher than that of AA type individuals. Therefore, the APAO1 gene core promoter polymorphism site g.-572A>G can be used to evaluate the resistance of cattle to ketosis.

Wherein, the dairy cow is a Chinese Holstein dairy cow.

The second aspect of the present invention provides the application of the above method in any of the following 1)-9):

1) Identify or assist in the identification of ketosis resistance of the cows to be tested;

2) Preparation of products to identify or assist in identifying the ketosis resistance of the cows to be tested;

3) Identify or assist in identifying the cows to be tested as ketosis-resistant cows;

4) Preparation of products to identify or assist in identifying the cows to be tested as ketosis-resistant cows;

5) Breeding of dairy cows;

6) Breeding breeds resistant to ketosis in dairy cows;

7) Preparation of products for breeding ketosis-resistant varieties of dairy cows;

8) Identify or assist in identifying the ketosis resistance traits of the cows to be tested;

9) Preparation of products for identifying or assisting in identifying traits of ketosis resistance in dairy cows.

Wherein, the dairy cow is a Chinese Holstein dairy cow.

In another specific implementation manner of the present invention, the application mode includes:

Extract blood DNA from different individual cows;

Identify individuals with molecular markers of ketosis resistance.

The molecule is labeled g.-572A>G.

Compared with the prior art, the beneficial effects of the above technical solution are:

1) The detection method of the above-mentioned technical scheme is original. According to the genetic characteristics of the occurrence of bovine ketosis, from the perspective of genome evolution and selection and disease susceptibility, the group of ketosis cattle is used as the reference group, and the healthy cattle group is used as the experiment. Group, integrating multiple genomic positive selection signal analysis techniques, using ketosis or healthy binary traits in dairy cows as the phenotype and β-hydroxybutyric acid content in the blood of dairy cows as the phenotype, using genome-wide association analysis methods and strategies , Efficient and accurate screening of key genes and molecular markers related to the occurrence of bovine ketosis, the method design is reasonable, and the detection method designed based on the key genes and markers has the characteristics of high accuracy and simple application and operation;

2) The above technical solution provides a single nucleotide polymorphism in the APOA1 gene that is related to ketosis resistance. By detecting the genotype of the locus, the ketosis resistance can be easily evaluated. The ketosis resistance provides an effective basis for evaluation and saves economic costs for livestock breeding enterprises.

3) Based on the confirmation of the expression differences of the above genes and sites, the reagents used to detect the APOA1 gene, the reagents used to detect the single nucleotide polymorphism site of the APOA1 gene and the primers can be used to prepare a kit for the detection of ketosis resistance in dairy cows. , Provides a convenient product for ketosis resistance in dairy cows, so it has good practical application value.

Description of the drawings

The accompanying drawings of the specification constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments and descriptions of the present invention are used to explain the present disclosure, and do not constitute an improper limitation of the present disclosure.

Figure 1 is a flow chart of the method for screening molecular markers for ketosis resistance in dairy cows in Example 1 of the present invention;

Fig. 2 shows the molecular marker APOA1 gene for dairy cow ketosis resistance screened by XPCLR forward selection signal analysis and PLINK whole genome association analysis in Example 1 of the present invention; where A is XPCLR forward selection signal analysis; B is PLINK whole genome Correlation Analysis;

Figure 3 is a schematic diagram of the APOA1 gene and its genome structure in Example 2 of the present invention, and the position information of SNP g.-572A>G in the genome.

Figure 4 shows the activity of promoters of different genotypes in the APOA1 gene in Example 3 of the present invention.

detailed description

It should be pointed out that the following detailed descriptions are all illustrative and are intended to provide further explanations for the application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate There are features, steps, operations, devices, components, and/or combinations thereof.

As described in the background art, the inventors found that although cattle have similar genetic backgrounds, there are significant differences in the phenotypes of β-hydroxybutyric acid content in their blood and whether they suffer from ketosis. It is speculated that their traits may be different. It is related to the single nucleotide polymorphism of the gene.

The design idea of the present invention is as follows: firstly, the candidate genes for ketosis resistance in dairy cows are screened through methods such as gene chip sequencing and whole gene association analysis. Secondly, the ketosis resistance candidate gene APOA1 was quantitatively analyzed, and the g.-572A>G site was identified in the core promoter region. Third, the concentration of β-hydroxybutyric acid in the blood of dairy cows with GG genotype at this locus was significantly lower than that of AA genotype, and the luciferase activity of the promoter of GG type was significantly higher than that of individuals of type AA, suggesting that the APOA1 gene is more abundant. The status g.-572A>G can be used as a marker for evaluating the resistance of dairy cows to ketosis.

In a specific embodiment of the present invention, there is provided a method for screening dairy cows for ketosis resistance genes, the method at least comprising:

Analyze DNA samples of healthy cows and cows suffering from ketosis based on SNP chip detection;

Analysis based on Fst, XPCLR and XPEHH genome selection signal analysis methods and GEMMA and PLINK genome-wide association analysis methods; screening SNPs and candidate genes that are significantly positively selected and significantly related to ketosis, and integrate gene function annotations Screen SNP molecular markers for ketosis resistance.

Wherein, the dairy cow is a Chinese Holstein dairy cow.

In another specific embodiment of the present invention, the method for screening dairy cows for ketosis resistance genes includes:

S1. Collection and DNA extraction of samples from healthy cows and cows suffering from ketosis;

S2. SNP chip detection and analysis;

S3. Genotype filling;

S4. Fst, XPCLR, XPEHH genome selection signal analysis;

S5. GEMMA, PLINK genome-wide association analysis;

S6. Screening candidate genes based on the screening strategy of candidate genes;

S7. Identify selected SNPs located in candidate genes;

Among them, steps S4 and S5 are in no order.

In another specific embodiment of the present invention, the specific method of step S1 includes:

S1.1 Determine the concentration of β-hydroxybutyric acid in the blood of dairy cows;

S1.2 Collect blood from healthy cows and cows suffering from ketosis, and extract DNA from blood tissues.

In another specific embodiment of the present invention, the specific method of step S2 includes:

S2.1 Use SNP chips to analyze DNA samples and perform genotyping;

S2.2 Filter the SNP data, and further analyze the remaining SNPs that meet the requirements;

S2.3 constructs a haplotype for each chromosome;

S2.4 Obtain 50K gene chip data in the database;

S2.5 uses the 150K gene chip as a reference to fill in the genotype of the 50K gene chip data.

In another specific embodiment of the present invention, the specific method of step S3 includes:

S3.1 Selection signal Fst and hapFLK genome scanning and local evolutionary tree construction: Fst analysis of all data obtained by dairy cow breeds;

S3.2 Estimate the XPCLR value between ketosis cows and healthy cow breeds;

S3.3 Estimate the XPEHH value between ketosis cows and healthy cow breeds.

In another specific embodiment of the present invention, the specific method of step S4 includes:

S4.1 uses GEMMA univariate linear mixed model, and conducts genome-wide association analysis with ketosis or healthy binary traits of dairy cows as the phenotype;

S4.2 Take the content of β-hydroxybutyric acid in the blood of dairy cows as the phenotype, use a multiple regression model, and use the parity and age of dairy cows as covariates to perform genome-wide association analysis;

S4.3 Calculate the significance P value based on the Benjamini and Hochberg correction method;

S4.4 Generate Manhattan chart of GWAS analysis;

S4.5 SNP annotations are retrieved in the UCSC database through Bedtools.

In another specific embodiment of the present invention, the specific method of step S5 includes:

S5.1 Select SNPs with strong selection signal and the most significant P value among Fst, XPCLR or XPEHH;

S5.2 Use the UCSC genome browser to retrieve the annotated Refseq genes in each selected region defined by the SNP;

S5.3 Select the top 1% SNP markers with the signal value obtained by each analysis method to locate the selected SNPs, limit the genes to within 500K bp upstream and downstream of each significant SNP, locate the positively selected genes; screen out simultaneous Genes identified by at least two analysis methods at the same time, and in at least one analysis method, the selected signal value or the P value of the significance test ranks the top 10 as important candidate genes;

S5.4 Use DVAID to perform functional analysis on the selected candidate genes, and apply Benjamini-Hochberg to perform multiple corrections to analyze which specific molecular functions and cellular components or biological pathways are enriched in.

In another specific embodiment of the present invention, the specific method of step S6 includes:

S6.1 Determine candidate genes and identify SNPs located in candidate genes that are selected;

S6.2 Perform correlation analysis on the identified SNPs and ketosis in dairy cows.

In another specific embodiment of the present invention, the cow ketosis resistance candidate gene includes APOA1; the SNP locus is g.-572A>G.

The site naming rules are as follows: the A base of the first ATG (translation initiation codon) of the APOA1 gene (NCBI: AC_000172.1) is named +1, and the downstream direction is +2, +3... ..., the upstream direction is -1, -2..., and the sites are respectively located at -572 in the upstream direction of the APOA1 gene.

In another specific embodiment of the present invention, the application of the above method in any of the following 1)-9) is provided:

1) Identify or assist in the identification of ketosis resistance of the cows to be tested;

2) Preparation of products to identify or assist in identifying the ketosis resistance of the cows to be tested;

3) Identify or assist in identifying the cows to be tested as ketosis-resistant cows;

4) Preparation of products to identify or assist in identifying the cows to be tested as ketosis-resistant cows;

5) Breeding of dairy cows;

6) Breeding ketosis-resistant breeds of dairy cows;

7) Preparation of products for breeding ketosis-resistant varieties of dairy cows;

8) Identify or assist in identifying the ketosis resistance traits of the cows to be tested;

9) Preparation of products for identifying or assisting in identifying traits of ketosis resistance in dairy cows.

Wherein, the dairy cow is a Chinese Holstein dairy cow.

In another specific implementation manner of the present invention, the application mode includes:

Extract blood DNA from different individual cows;

Identify individuals with molecular markers of ketosis resistance.

The molecular label is g.-572A>G;

The primers for amplifying gene fragments containing molecular markers include SEQ ID NO.1 and SEQ ID NO.2.

The content of the present invention will be further described below in conjunction with the embodiments, but it is not a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions in the following examples are usually carried out in accordance with conventional conditions.

Example 1 Screening of Candidate Genes for Bovine Ketosis Resistance

1. Collection of blood samples from Chinese Holstein cows

The pre-screening of cattle in the Shandong Province DHI database was carried out, and the DHI information was complete. 95 cattle with ketosis and 95 healthy cattle, a total of 190 cattle, these 190 cattle are evenly distributed in age and parity, and have been in the past. Within 7 to 14 days of the lactation period, at least two lactation periods have reported ketosis or health.

The blood is collected from the caudal vein of the cow, and the principle of vacuum negative pressure anticoagulation is used to collect the blood of the cow into a 5ml vacuum blood collection tube. During the first 7 to 14 days of milk production, the blood ketone meter was used to measure the concentration of β-hydroxybutyric acid in the blood of 190 cows, and a drop of blood from the cows at the early stage of milk production was dripped onto the chip test strip matched with the blood ketone meter. , Insert the blood ketone meter, wait for 10 seconds for the analysis and calculation of the instrument, read and record the concentration of β-hydroxybutyric acid in the blood, and repeat the measurement experiment twice.

2. Use Illumina BovineHD 150KSNP chip for genotyping

The blood cards of 190 cattle blood DNA samples qualified and titrated were sent to the gene chip sequencing company, and the DNA of 190 cattle was genotyped and data assembled using the American Illumina Bovine 150K gene chip to obtain genotype data. Use Plink 1.9 software to perform quality control on the 150K gene chip data of 190 cattle. To ensure that the genotype data filled in the reference gene chip are not missing and phased, samples with a genotype missing rate greater than 0 and samples with a sample missing rate greater than 0 Get rid of.

3. Genotype filling

Download Huang et al.'s (2019) ketosis-related gene chip data containing 2488 cattle and 50K SNP from the NAGRP database. Using the two-step genotype filling method of Fimpute and Beagle software, with the 150K gene chip of 190 cattle as a reference, the 50K gene chip of 2488 cattle in the NAGRP database was used for genotype filling.

4. Fst, XPEHH, XPCLR genome selection signal analysis

The ketosis group was used as the reference group, and the healthy group was used as the experimental group, that is, the positive selection group. Three methods suitable for disease-control positive selection signal analysis: Fst, XPEHH, and XPCLR are used for selection signal analysis. Use Vcftool software for Fst selection signal analysis and calculate Fst value; use Selscan software for XPEHH forward selection signal analysis; use XPCLR software for XPCLR selection signal analysis.

5. GEMMA, PLINK genome-wide association analysis

Taking dairy cows suffering from ketosis or healthy binary traits as the phenotype, using the general linear model of GEMMA software, a genome-wide association analysis was performed on 115787 markers of 2678 cows after genotype filling; β-hydroxybutyric acid in blood of dairy cows The content was used as the phenotype, and the genome-wide association analysis was performed on 140,668 markers of 190 cattle using the multiple regression model of Plink software.

6. Subject to positive selection genetic variation and candidate gene screening

Use Fst, XPCLR, XPEHH selection signal analysis methods and GEMMA, PLINK whole genome association analysis methods to screen SNPs related to ketosis, with the selection signal top 1% as the threshold, and P＜0.05 as the threshold for the whole genome association analysis. There are 98 candidate genes related to ketosis resistance, including the APOA1 gene.

Example 2 Identification of SNP in the bovine ketosis resistance gene APOA1

1. The expression analysis of APOA1 gene in healthy and ketotic dairy cows

Quantitative detection of blood samples from healthy and ketotic dairy cows using fluorescent quantitative PCR showed that the expression level of APOA1 gene in ketotic dairy cows was significantly higher than that in healthy dairy cows (P<0.05).

2. SNP sequencing identification of bovine ketosis resistance gene APOA1

(1) Choose 95 healthy cattle and 95 ketosis cattle to extract blood DNA.

(2) Screen the polymorphic sites in the 5'flanking region of CPT1A gene. Design a pair of amplification primers:

F: 5'-CTCTGCTGCCTTTGTGAAG-3' (SEQ ID NO.1);

R: 5'-GCACCCTCTACTCACTCCAT-3' (SEQ ID NO. 2).

Subsequently, PCR amplification was performed, and the PCR products were detected by 1% agarose gel electrophoresis. Direct sequencing of the amplified product revealed that APOA1 detected a SNP at position -572 (g.-572A>G).

(3) Use SAS 9.4 least squares model combined with general linear model to analyze the correlation between SNP g.-572A>G of APOA1 gene and the content of β-hydroxybutyric acid in blood of dairy cows, and β-hydroxybutyrate in blood of dairy cows of GG genotype The acid concentration was significantly lower than the AA genotype.

Example 3 Detection of promoter activity of different genotypes of APOA1 gene

In order to further study the promoter activity of individuals with different genotypes in the promoter region, a 647bp promoter fragment of the APOA1 gene was amplified, and promoter fragments with genotypes of GG and AA were selected to construct pGL3-basic-GG and The promoter vector of pGL3-basic-AA type was transfected into HepG2 cells, and its luciferase activity was measured. The results showed that the luciferase activity of the GG type promoter of APOA1 gene was significantly higher than that of the AA type individual (P<0.05).

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than limiting them. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A method for screening dairy cattle ketosis resistance genes, characterized in that the method at least comprises:

   Analyze DNA samples of healthy cows and cows suffering from ketosis based on SNP chip detection;

   Analysis based on Fst, XPCLR and XPEHH genome selection signal analysis methods and GEMMA and PLINK genome-wide association analysis methods; screening SNPs and candidate genes that are significantly positively selected and significantly associated with ketosis, and integrate gene function annotations Screen SNP molecular markers for ketosis resistance.
2. The method of claim 1, wherein the method comprises:

   S1. Collection and DNA extraction of samples from healthy cows and cows suffering from ketosis;

   S2. SNP chip detection and analysis;

   S3. Genotype filling;

   S4. Fst, XPCLR, XPEHH genome selection signal analysis;

   S5. GEMMA, PLINK genome-wide association analysis;

   S6. Screening candidate genes based on the screening strategy of candidate genes;

   S7. Identify selected SNPs located in candidate genes;

   Among them, steps S4 and S5 are in no order.
3. The method according to claim 2, wherein the specific method of step S1 comprises:

   S1.1 Determine the concentration of β-hydroxybutyric acid in the blood of dairy cows;

   S1.2 Collect blood from healthy cows and cows suffering from ketosis, and extract DNA from blood tissues.
4. The method according to claim 2, wherein the specific method of step S2 includes:

   S2.1 Use SNP chips to analyze DNA samples and perform genotyping;

   S2.2 Filter the SNP data, and further analyze the remaining SNPs that meet the requirements;

   S2.3 constructs a haplotype for each chromosome;

   S2.4 Obtain 50K gene chip data in the database;

   S2.5 uses the 150K gene chip as a reference to fill in the genotype of the 50K gene chip data.
5. The method according to claim 2, wherein the specific method of step S3 includes:

   S3.1 Selection signal Fst and hapFLK genome scanning and local evolutionary tree construction: Fst analysis of all data obtained by dairy cow breeds;

   S3.2 Estimate the XPCLR value between ketosis cows and healthy cow breeds;

   S3.3 Estimate the XPEHH value between ketosis cows and healthy cow breeds.
6. The method according to claim 2, wherein the specific method of step S4 comprises:

   S4.1 uses GEMMA univariate linear mixed model to conduct genome-wide association analysis with dairy cows suffering from ketosis or healthy binary traits as the phenotype;

   S4.2 Take the content of β-hydroxybutyric acid in the blood of dairy cows as the phenotype, use the multiple regression model, and use the parity and age of the dairy cows as covariates to conduct genome-wide association analysis;

   S4.3 Calculate the significance P value based on the Benjamini and Hochberg correction method;

   S4.4 Generate Manhattan chart of GWAS analysis;

   S4.5 SNP annotations are retrieved in the UCSC database through Bedtools.
7. The method according to claim 2, wherein the specific method of step S5 comprises:

   S5.1 Select SNPs with strong selection signal and the most significant P value among Fst, XPCLR or XPEHH;

   S5.2 Use the UCSC genome browser to search for the annotated Refseq genes in each selected region defined by the SNP;

   S5.3 Select the top 1% SNP markers with the signal value obtained by each analysis method to locate the selected SNPs, limit the genes to within 500K bp upstream and downstream of each significant SNP, locate the positively selected genes; screen out simultaneous Genes identified by at least two analysis methods at the same time, and in at least one analysis method, the selected signal value or the P value of the significance test ranks the top 10 as important candidate genes;

   S5.4 Use DVAID to perform functional analysis on the selected candidate genes, and apply Benjamini-Hochberg to perform multiple corrections to analyze which specific molecular functions and cellular components or biological pathways the genes are enriched in.
8. The method according to claim 2, wherein the specific method of step S6 comprises:

   S6.1 Determine candidate genes and identify SNPs located in candidate genes that are selected;

   S6.2 Perform correlation analysis on the identified SNPs and ketosis in dairy cows.
9. The method of claim 2, wherein the candidate genes for ketosis resistance in dairy cows include APOA1; and the SNP locus is g.-572 A>G.
10. The application of the method according to any one of claims 1-9 in any one of the following 1)-9):

    1) Identify or assist in the identification of ketosis resistance of the cows to be tested;

    2) Preparation of products to identify or assist in identifying the ketosis resistance of the cows to be tested;

    3) Identify or assist in identifying the cows to be tested as ketosis-resistant cows;

    4) Preparation of products to identify or assist in identifying the cows to be tested as ketosis-resistant cows;

    5) Breeding of dairy cows;

    6) Breeding breeds resistant to ketosis in dairy cows;

    7) Preparation of products for breeding ketosis-resistant varieties of dairy cows;

    8) Identify or assist in identifying the ketosis resistance traits of the cows to be tested;

    9) Preparation of products to identify or assist in the identification of ketosis resistance traits of dairy cows;

    Preferably, the application method includes:

    Extract blood DNA from different individual cows;

    Identify individuals with molecular markers of ketosis resistance;

    Further preferably, the molecular label is g.-572 A>G;

    The primers for amplifying gene fragments containing molecular markers include SEQ ID NO.1 and SEQ ID NO.2.

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