CN113416789B - Molecular marker related to concentration of beta-hydroxybutyric acid in milk cow blood and application thereof - Google Patents
Molecular marker related to concentration of beta-hydroxybutyric acid in milk cow blood and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cow ketosis resistance breeding, and relates to a molecular marker related to the concentration of beta-hydroxybutyric acid in cow blood and an application thereof, wherein the molecular marker comprises an SNP site g.66266G > T located on a UBA6 gene. The invention provides a molecular marker related to the concentration of beta-hydroxybutyric acid in milk cow blood and application thereof, provides SNP sites related to the concentration of beta-hydroxybutyric acid in milk cow blood in UBA6 gene and additive genetic effect thereof, and has good practical application value.
Description
Technical Field
The invention belongs to the technical field of cow ketosis resistance breeding, and relates to a molecular marker related to the concentration of beta-hydroxybutyric acid in cow blood and application thereof.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Ketosis (ketosis) is a nutritional metabolic disease occurring in perinatal cows based on the major pathologies of energy negative balance, fat mobilization, hyperketonemia, hypoglycemia and disorders of hepatic lipid metabolism. Ketosis occurs frequently in high-producing cows, and has a negative effect on the health, reproductive capacity and milk yield of the cows. In the early lactation period of high-yield cows, the milk yield is increased, a large amount of lactose is synthesized, so that glucose in a body is mobilized to synthesize lactose, and the sugar ingested by the body is insufficient, so that the energy is deficient, a large amount of fatty acid in the body is promoted to be decomposed, and the deficient energy is compensated. At the moment, the fat reserve of the body is excessively mobilized, a large amount of non-esterified fatty acid is oxidized, so that a large amount of ketone bodies are generated in the liver of the cow, but the rate of metabolizing the non-esterified fatty acid by the body is limited, so that the ketone bodies in the blood, urine and milk of the cow are accumulated, and the content of the ketone bodies is increased. The ketone body is an intermediate product generated when fatty acid is decomposed and oxidized in liver cells, and comprises three components of acetoacetic acid, beta-hydroxybutyric acid (BHB) and acetone. The BHB concentration in blood was used as a gold standard indicator of ketosis, and a threshold of 1.2mmol/L was used to identify ketosis cows (Van der Drift et al, 2012).
Ketosis is resistant, but its heritability is low, between 0.02 and 0.06, and it is difficult to achieve effects by direct selection based on phenotype (Koeck et al, 2013; Parker Gaddis et al, 2014). BHB is an indicative trait of ketosis with moderate heritability of 0.07-0.40(Oikonomou et al, 2008; Jamrozikik et al, 2016). Therefore, indirect selection of ketosis is more effective by using BHB as an index trait, and a better genetic gain can be obtained than direct selection according to ketosis. Huang et al (2019) use the concentration of beta-hydroxybutyrate in cow blood as a defining standard, and carry out whole genome association analysis by adopting binary traits of a healthy group and a ketosis group to obtain candidate genes related to ketosis, such as GCH 1. Yan et al (2020) select 4 healthy cattle and 8 ketosis cattle according to the concentration of beta-hydroxybutyric acid in blood, perform transcriptome sequencing and whole genome correlation analysis, and newly identify 5 ketosis candidate genes such as GRINA. Whether the candidate genes affect ketosis resistance remains to be verified. Screening and identifying candidate genes and molecular markers related to the concentration of beta-hydroxybutyric acid in the blood of the dairy cattle is the basis for developing the application of the dairy cattle ketosis resistance molecular breeding.
Disclosure of Invention
UBA6 is ubiquitin activating enzyme, participates in ubiquitination modification, and plays an important role in embryonic development, nervous system development and function, and metabolic activity. Ubiquitination, as a post-translational modification of proteins, can play a regulatory role in liver lipid metabolism. Ubiquitination modification plays an important regulatory role in the activation of adenylate activated protein kinase (AMPK). The ubiquitination degradation mechanism is a function that AMPK increases the activity of endoplasmic reticulum anchoring protein through phosphorylation, and further inhibits liver lipid synthesis, and is a mechanism that protein posttranslational modification regulates fatty acid synthesis through a ubiquitination degradation pathway.
The inventor discovers for the first time that UBA6 genetic variation is obviously related to the blood BHB concentration of the milk cow and the breeding value of a ketosis genome, has an additive genetic effect and provides important evidence for ketosis resistance molecule breeding.
In order to screen and identify candidate genes and molecular markers related to the concentration of beta-hydroxybutyric acid in milk cow blood and provide richer basis for molecular breeding of ketosis resistant cattle, the disclosure provides molecular markers related to the concentration of beta-hydroxybutyric acid in milk cow blood and application thereof, provides SNP sites related to the concentration of beta-hydroxybutyric acid in milk cow blood in UBA6 gene and additive genetic effect thereof, and has good practical application value.
Specifically, the invention is realized by the following technical scheme:
in one or more embodiments of the invention, the molecular marker related to the concentration of beta-hydroxybutyrate in cow blood comprises the g.66266G > T SNP site located on the UBA6 gene.
In the region of intron 20 of the UBA6 gene, 1 SNP g.66266G > T was identified, the molecular marker being located at position 417 of the sequence indicated by SEQ ID NO.3, with the nucleotides G or T. Tests and correlation analysis prove that the concentration of beta-hydroxybutyric acid in blood of UBA6 gene TT type dairy cattle is obviously lower than that of GG gene.
In one or more embodiments of the invention, the primer combination is used for detecting the molecular marker related to the concentration of the beta-hydroxybutyric acid in the blood of the dairy cow.
The skilled in the art can design suitable specific primers based on specific site information, and can realize the detection of the SNP molecular markers, therefore, any primer combination for detecting the SNP molecular markers of the invention belongs to the protection scope of the invention.
In one or more embodiments of the invention, the primer combination is used for preparing a kit for identifying the ketosis resistance of the dairy cow. The screening of cow with ketosis resistance can be realized by those skilled in the art by using the primer combination, and the design of a kit for identifying the ketosis resistance of cow aiming at the primer combination is also within the protection scope of the invention.
In one or more embodiments of the invention, the application of the molecular marker related to the concentration of beta-hydroxybutyric acid in milk cow blood in the assistant selection breeding of milk cow molecular markers comprises the following steps: collecting milk cow blood DNA, and carrying out SNP typing detection on milk cow DNA samples aiming at the SNP loci of claim 1; and carrying out genotyping detection result and blood beta-hydroxybutyric acid concentration correlation analysis.
In one or more embodiments of the invention, a method for screening ketosis resistant cows comprises extracting genomic DNA of cows, determining the 417 th base polymorphism of the UBA6 gene of cows, and predicting the ketosis resistance of cows according to the 417 th haplotype combination of cows. Because the blood concentration of beta-hydroxybutyric acid of the TT genotype dairy cattle individual is obviously lower than that of the GG genotype dairy cattle individual (P < 0.05). Therefore, screening for a favorable genotype TT with a low blood β -hydroxybutyrate concentration phenotype can be used to aid in the selection of dairy individuals with high ketosis resistance. Therefore, when the haplotype combination is TT type, the concentration of beta-hydroxybutyric acid in the blood of the dairy cow is low, and the ketosis resistance is strong.
In one or more embodiments of the invention, the molecular marker related to the concentration of beta-hydroxybutyrate in cow blood or the primer combination is applied to cow breeding, genetic improvement or marker-assisted selection. By exploring and obtaining the SNP locus related to the concentration of the beta-hydroxybutyric acid in the UBA6 gene, the cow with ketosis resistance can be selected based on the SNP locus, and then the cow breeding, genetic improvement and marker assistance are carried out, so that the method has better application value.
In one or more embodiments of the invention, the use of the UBA6 gene for screening ketosis resistant cows.
In one or more embodiments of the invention, a primer for screening SNP sites of UBA6 gene is designed according to UBA6 gene sequence AC _000163.1(85050203..85137078) and a pair of primers UBA6-F (SEQ ID NO.1) and UBA6-R (SEQ ID NO. 2).
In one or more embodiments of the invention, the molecular marker related to the concentration of beta-hydroxybutyric acid in the blood of the dairy cow is used for identifying and assisting in identifying a dominant individual with low concentration of beta-hydroxybutyric acid in the blood of the dairy cow or preparing a product for identifying and assisting in identifying the ketosis resistance of the dairy cow.
One or more embodiments of the present invention have the following advantageous effects:
(1) according to the method, a 50K SNP chip of a cow is used for genotyping the Holstein cow, a new candidate gene UBA6 which influences the ketosis of the cow is screened out through whole genome association analysis, and a new target point is provided for detecting the ketosis resistance gene of the cow.
(2) The disclosure provides SNP loci related to the concentration of beta-hydroxybutyric acid in milk cow blood in UBA6 gene, which can be used for early breeding of milk cows by analyzing the genotype of the SNP loci and indirectly selecting milk cow ketosis resistant individuals.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: the UBA6 gene was screened in the Manhattan plot of the GEMMA genome-wide association analysis in example 1.
FIG. 2: the structural schematic diagram of UBA6 gene on genome in example 2, and the positional information of SNP g.66266G > T.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
A method for screening molecular markers related to the concentration of beta-hydroxybutyric acid comprises the following steps: the SNP chip is used for sequencing and genotyping, a genome breeding value is estimated according to genotyping data, the genotyping data and the ketosis genome breeding value are subjected to whole genome correlation analysis, candidate genes and SNP sites which are obviously related to ketosis are screened, and molecular markers related to the concentration of beta-hydroxybutyric acid are screened.
The whole genome association analysis specifically comprises: genome-wide association analysis was performed using a mixed linear model of GEMMA software, with ketosis genome breeding values as phenotypes.
The method for screening candidate genes which are significantly related to ketosis comprises the following steps: according to the whole genome association analysis result, a remarkable SNP locus is located, and a candidate gene UBA6 is anchored according to the position of the SNP locus on the chromosome downstream.
The process of screening SNP sites includes: screening healthy cattle and ketosis cattle according to the concentration of beta-hydroxybutyric acid in blood, and preserving DNA in the blood at low temperature; and (3) carrying out PCR amplification by taking DNA in blood as a template, directly carrying out sequencing analysis on a PCR amplification product, comparing a sequencing result with a bovine UBA6 gene sequence, and screening out the SNP locus.
The healthy cattle screening standard is that the concentration of beta-hydroxybutyric acid in blood is below 0.4mmol/mL, and the ketosis cattle screening standard is that the concentration of beta-hydroxybutyric acid in blood is above 1.2 mmol/mL.
The correlation analysis with β -hydroxybutyrate concentration included: genotyping healthy and ketotic dairy individuals using primers UBA6-F and UBA 6-R; by using software, different genotypes of the UBA6 gene at the SNP locus g.66266G > T and the concentration of beta-hydroxybutyric acid in the blood of the dairy cow are subjected to correlation analysis. The correlation analysis result shows that the blood concentration of beta-hydroxybutyric acid of the TT genotype dairy cow individual is obviously lower than that of the GG genotype dairy cow individual (P < 0.05). Therefore, screening of the favorable genotype TT of the phenotype with low blood beta-hydroxybutyrate concentration can be used for assisting in selecting dairy cow individuals with high ketosis resistance.
The present invention is described in further detail below with reference to specific examples, which should be construed as illustrative rather than restrictive.
Example 1
Screening candidate genes affecting dairy cow ketosis by using technologies such as chip sequencing and whole gene correlation analysis
1. Sequencing and genotyping Using bovine 50K SNP chips
(1) Collecting hair follicle samples of 2709 Holstein cows in domestic large-scale dairy farms, extracting DNA in hair follicles, and sending the DNA to Illumina company for genotyping by using a cow 50KSNP chip.
(2) Genotype information of SNP sites contained in the 50K chip of each individual bovine was obtained.
(3) And estimating and obtaining a ketosis genome breeding value of 2709 dairy cow individuals by utilizing a genome evaluation system according to the genotype data.
2. Screening candidate genes affecting dairy cow ketosis resistance by using whole genome correlation analysis method
(1) Using a mixed linear model of GEMMA software, the genome-wide association analysis was performed on 47843 SNP markers of 2709 cows with the ketosis genome breeding value as a phenotype. The P value was corrected using the FDR method.
(2) According to the whole genome association analysis result, 14 significant SNP sites are located. These 14 SNP sites were anchored to the candidate gene UBA6 (FIG. 1) at a position 100kb downstream on the chromosome.
Example 2
Identifying UBA6 gene SNP locus, and screening molecular markers related to milk cow blood beta-hydroxybutyric acid concentration
1. Collecting blood samples of healthy and ketosis cattle, and measuring concentration of beta-hydroxybutyric acid in blood
(1) Collecting blood samples of cows 21 days after the large-scale cattle farm, measuring the ketone body concentration by using a cow blood ketone body detector, and screening 95 healthy cows and 95 ketosis cows according to the concentration of beta-hydroxybutyric acid in blood. The healthy cattle screening standard is that the concentration of beta-hydroxybutyric acid in blood is below 0.4mmol/mL, and the ketosis cattle screening standard is that the concentration of beta-hydroxybutyric acid in blood is above 1.2 mmol/mL.
(2) Extracting DNA from blood by using a blood DNA extraction kit, and storing at-20 ℃ for later use.
Identification of SNP sites of UBA6 Gene
A pair of primers UBA6-F (SEQ ID NO.1) and UBA6-R (SEQ ID NO.2) are designed according to a bovine UBA6 gene sequence AC _000163.1(85050203..85137078), and PCR amplification is carried out by taking DNA in blood as a template. The sequence after PCR amplification is shown as SEQ ID NO. 3. The PCR amplification product is directly subjected to sequencing analysis, the sequencing result is compared with the bovine UBA6 gene sequence, and 1 SNP site g.66266G > T is found in the intron 20 region (figure 2).
Genotyping of SNP site of UBA6 Gene, correlation with concentration of beta-hydroxybutyric acid in cow blood and analysis of additive Effect
(1) 189 healthy and ketotic dairy individuals were genotyped using primer pairs UBA6-F and UBA 6-R. And performing correlation analysis on different genotypes of the SNP site g.66266G > T of the UBA6 gene and the concentration of beta-hydroxybutyric acid in the blood of the dairy cow by using SAS 8.0 software. The correlation analysis result shows that the blood concentration of beta-hydroxybutyric acid of the TT genotype dairy cow individual is obviously lower than that of the GG genotype dairy cow individual (P < 0.05).
(2) Additive effect of the gene:
homozygote mean value: m ═ BHB (BHB)GG-BHBTT)/2＝(1.82+0.94)/2＝1.38(mmol/L)
Gene additive effect: alpha ═ BHBTT-m＝0.94-1.38＝-0.44(mmol/L)
Therefore, the beneficial genotype for screening the phenotype of low blood beta-hydroxybutyrate concentration is TT, the additive effect is-0.44 mmol/L, and the method can be used for assisting in selecting dairy cow individuals with high ketosis resistance.
TABLE 1 correlation analysis of different genotypes of SNP loci of UBA6 genes of dairy cows and blood beta-hydroxybutyric acid concentration
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. The application of the SNP locus related to the concentration of beta-hydroxybutyric acid in the blood of the dairy cow in the molecular marker-assisted selective breeding of the dairy cow is characterized by comprising the following steps: collecting milk cow blood DNA, and carrying out SNP typing detection on a milk cow DNA sample aiming at an SNP locus related to the concentration of beta-hydroxybutyric acid in the milk cow blood; carrying out genotyping detection result and blood beta-hydroxybutyric acid concentration correlation analysis; screening the favorable genotype TT of the low blood beta-hydroxybutyrate concentration phenotype;
wherein the SNP locus related to the concentration of the beta-hydroxybutyric acid in the blood of the cow is the SNP locus g.66266G > T on the intron 20 region of the UBA6 gene.
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| CN105200146A (en) * | 2015-10-27 | 2015-12-30 | 湖北省农业科学院畜牧兽医研究所 | SNP genetic marker correlated to pig fat deposition property and application |
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| CN111485027A (en) * | 2020-06-09 | 2020-08-04 | 山东省农业科学院奶牛研究中心 | Method for screening dairy cow ketosis resistance molecular marker and application thereof |
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| CN105200146A (en) * | 2015-10-27 | 2015-12-30 | 湖北省农业科学院畜牧兽医研究所 | SNP genetic marker correlated to pig fat deposition property and application |
| CN107267605A (en) * | 2017-06-13 | 2017-10-20 | 甘肃民族师范学院 | The SNP marker related to china holstein cowses reproductive trait and its application |
| CN111485027A (en) * | 2020-06-09 | 2020-08-04 | 山东省农业科学院奶牛研究中心 | Method for screening dairy cow ketosis resistance molecular marker and application thereof |
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