A method for evaluating lactose content in cow milk
The combination of DNA direct sequencing and PCR-RFLP method to detect the SNP site of the LBP gene, solving the problem of evaluating the content of dairy nugget in the prior art, achieving a simple and reliable evaluation of the content of dairy nugget, reducing costs, and is suitable for molecular breeding of dairy cow production performance.
Patent Information
- Application Number
- CN202210750602.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-06-29
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2042-06-29
AI Technical Summary
There is a lack of effective methods in the prior art to evaluate the lactose content in dairy cow milk, and high-throughput sequencing equipment is expensive and complex to operate, and is not suitable for widespread applications in ordinary molecular laboratories.
The technology of combining direct DNA sequencing and PCR-RFLP method was used to detect the polymorphism of the SNP site of the LBP gene. The RFLP method was used to detect the enzyme cutting products through agarose electrophoresis to identify the genotype of the SNP site on the LBP gene of dairy cows.
A molecular marker I2-1095 G>A, which affects the trait of dairy nugget, was discovered, and a simple and reliable method for evaluating the content of dairy nugget was established, which reduced costs and was suitable for molecular breeding of dairy cow production performance.
Smart Images
Figure CN114990234B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular breeding of dairy cows, and more particularly to a method for evaluating the lactose content in dairy cow milk. Background Art
[0002] Single nucleotide polymorphisms (SNPs), DNA sequence polymorphisms caused by single base transitions, transversions, insertions, or deletions, are the primary source of genetic polymorphism. SNPs, third-generation polymorphic markers, are an important indicator of genetic variation within animal strains and have been widely used in population genetics research. Classic SNP detection methods based on gel electrophoresis include restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), and allele-specific PCR (AS-PCR). High-throughput sequencing is a rapidly developing SNP detection method in recent years. It includes DNA sequencing, DNA chip detection, restriction-site associated DNA sequence (RAD-Seq), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS), denaturing high-performance liquid chromatography (DHP LC), and more. High-throughput sequencing is prohibitive for widespread research in general molecular laboratories due to the high cost of the equipment and the technical expertise required. Among traditional research methods, SSCP (Sensory Specific Clustering Score) is prone to false negative results and is complex and time-consuming, making it a suboptimal SNP detection method.
[0003] Dairy herd improvement (DHI) tests the production performance of each cow, scientifically and digitally analyzes the quantitative traits of dairy cows, takes both group and individual levels into consideration, improves pasture management and enhances economic benefits.
[0004] The DHI report is broadly divided into three areas: nutritional indicators, reproductive indicators, and milk quality and parlor management indicators. Nutritional indicators include daily milk production, fat-to-egg ratio, endurance, peak milk, peak day, corrected milk, 305-day milk production, and adult milk equivalent. Reproductive indicators include days of lactation, calving interval, and urea nitrogen. Milk quality and parlor management indicators include somatic cell count, milk fat percentage, milk protein percentage, and the rate of latent mastitis. Basic test indicators include daily milk production, milk fat percentage, milk protein percentage, somatic cell count, lactose percentage, and total solids percentage.
[0005] (1) Daily milk production: The total milk production of lactating dairy cows on the test day, in kilograms, reflecting the actual milk production level of the cows.
[0006] (2) Milk fat content: the percentage of fat in milk.
[0007] (3) Milk protein rate: the percentage of protein contained in milk.
[0008] (4) Lactose rate: the percentage of lactose contained in milk.
[0009] (5) Somatic cell count (SCC): The content of white blood cells per milliliter of milk, including lymphocytes, macrophages, polymorphonuclear neutrophils, etc., is an important indicator of the health of the cow's udder.
[0010] Lactose is naturally found only in mammalian milk. It is one of the three main nutrients in milk and accounts for a quarter of the total calories in milk. Normal milk contains 4.6%-5.0% lactose, which affects milk quality, helps maintain a stable intestinal flora, and can also indicate the development of certain pathologies. For example, when a cow is suffering from tuberculosis or mastitis, the lactose content in its milk drops dramatically. Lactose is composed of glucose and galactose, with galactose affecting brain development by binding to ceramides. Lactose also affects milk production by affecting the amount of water that enters mammary cells. Research on lactose regulation can help address lactose intolerance. Reducing lactose concentration in milk can improve patients' lactose intolerance, significantly reducing treatment costs compared to drug-mediated treatments.
[0011] Chinese patents have disclosed molecular markers associated with milk protein content and 305-day milk production in Holstein cows; molecular markers associated with total milk production and peak milk production in Holstein cows; and molecular markers associated with milk protein content and somatic cell count in Holstein cows. Currently, SNP markers associated with lactose content in dairy cows and their applications have not been disclosed.
[0012] Lipopolysaccharide-binding protein (LPBP), named for its ability to readily bind to lipopolysaccharides (LPS), is a plasma protein produced in the liver and present in serum. LBP controls the LPS response by binding to LPS to form a complex. CD14 further binds to the LBP / LPS complex, activating Toll-like receptor 4 (TLR4), which releases proinflammatory cytokines and chemokines, inducing an inflammatory response. However, LBP has not been associated with dairy cow performance. Summary of the Invention
[0013] The present invention aims to overcome the shortcomings of the prior art and provide a method for assessing lactose content in cow milk. The RFLP method is simple and can directly determine the individual genotype based on the results of agarose electrophoresis. The present invention utilizes a combination of direct DNA sequencing and PCR-RFLP to detect the effect of SNP polymorphisms in the LBP gene on the lactose trait of Chinese Holstein cows.
[0014] This study used dairy cows as experimental subjects, analyzing single nucleotide polymorphisms (SNPs) of the LBP gene through direct DNA sequencing and PCR-RFLP to explore the correlation between LBP gene polymorphisms and dairy cow production traits. Using whole-genome DNA from the cows to be tested as a template and primer I2 as a primer, PCR was performed to amplify a partial sequence from the second intron of the LBP gene. The PCR product was treated with the restriction endonuclease Afl II, and the digested product was detected by agarose gel electrophoresis. The genotype of the SNP site in the cow LBP gene was identified based on the band length. The results showed that a SNP site was found in this population, located at base 1095 of the second intron of the LBP gene (i.e., I2-1095 G>A). Correlation analysis revealed that different genotypes of this SNP site were highly significantly associated with lactose production traits. Individuals with the genotype GA had significantly higher lactose production traits than those with the GG genotype. This site can be used as a molecular marker for dairy cow selection and breeding.
[0015] The first object of the present invention is to provide a reagent for evaluating the lactose content in cow's milk.
[0016] The second object of the present invention is to provide a use of the reagent in evaluating the lactose content in cow's milk or in preparing a kit for evaluating the lactose content in cow's milk.
[0017] The third object of the present invention is to provide a method for evaluating the lactose content in cow's milk.
[0018] The fourth object of the present invention is to provide a kit for evaluating the lactose content in cow's milk.
[0019] A fifth object of the present invention is to provide the use of one or more of the reagent, the method, or the kit according to claim 6 in molecular breeding of lactose content in cow milk.
[0020] In order to achieve the above object, the present invention is implemented through the following technical solutions:
[0021] The present invention claims a reagent for evaluating the lactose content in cow milk, wherein the reagent is used to detect the genotype of ARS-UCD1.2 version NC_037340.1:67219155, wherein the lactose content in the milk of individual cows with the genotype GA is higher than that in the milk of individual cows with the genotype GG.
[0022] The invention also claims the use of the reagent in evaluating the lactose content in cow's milk or preparing a kit for evaluating the lactose content in cow's milk.
[0023] A method for evaluating the lactose content in cow milk is provided, wherein the genotype of ARS-UCD1.2 version NC_037340.1:67219155 is detected, and the lactose content in the milk of individual cows with the genotype GA is higher than the lactose content in the milk of individual cows with the genotype GG.
[0024] Preferably, the primers with nucleotide sequences as shown in SEQ ID NOs: 1-2 are used to detect the genotype of ARS-UCD1.2 version NC_037340.1: 67219155, and the lactose content in the milk of individual cows with the genotype GA is higher than that in the milk of individual cows with the genotype GG.
[0025] Upstream primer sequence: 5′-TGAAGGCTTGACAACGGAG-3′ (SEQ ID NO: 1);
[0026] Downstream primer sequence: 5'-AGACCCACAGAAGAGGCATC-3' (SEQ ID NO: 2).
[0027] The PCR system was as follows: Green Taq Mix 10 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, total volume 20 μL.
[0028] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.
[0029] PCR products were detected by 1.5% agarose gel electrophoresis at 120 V, 400 mA, and 15 min. Sequencing was performed after confirming that the bands were single, of the correct length, and without specific bands.
[0030] More preferably, the PCR product of the primers is digested with endonuclease Afl II. After digestion, individuals with a single band of 624 bp in electrophoresis are GG genotype individuals, individuals with three bands of 624 bp, 234 bp and 390 bp are GA genotype individuals, and individuals with two bands of 234 bp and 390 bp are AA genotype individuals.
[0031] PCR products were examined by 1.5% agarose gel electrophoresis at 120 V, 400 mA, and 15 min. After confirming the presence of a single, correct-length band and the absence of specific bands, the product was digested with Afl II using the following digestion system: 14 μL of PCR product, 0.2 μL of Afl II, 2 μL of 10× rCutSmart Buffer, and 3.8 μL of ddH2O, for a total volume of 10 μL.
[0032] The reaction system was incubated in a 37°C incubator for 2 h. 5 μl of the digested product was subjected to 2.5% agarose gel electrophoresis to detect the band length for genotyping. Electrophoresis conditions: 80 V, 400 mA, 10 min.
[0033] Also, a kit for evaluating the lactose content in cow's milk contains the reagent.
[0034] Preferably, the reagent is a primer having a nucleotide sequence as shown in SEQ ID NO: 1-2.
[0035] Upstream primer sequence: 5′-TGAAGGCTTGACAACGGAG-3′ (SEQ ID NO: 1);
[0036] Downstream primer sequence: 5'-AGACCCACAGAAGAGGCATC-3' (SEQ ID NO: 2).
[0037] More preferably, it further contains PCR reagents, including but not limited to Green Taq Mix.
[0038] More preferably, it further contains endonuclease Afl II and 10×rCutSmart Buffer.
[0039] The invention further claims the use of one or more of the reagent, the method, or the kit in molecular breeding of lactose content in cow milk.
[0040] Compared with the prior art, the present invention has the following beneficial effects:
[0041] Using dairy cows as a research subject, a molecular marker related to lactose production in dairy cows, I2-1095G>A, was discovered. It was found that individuals with the GA genotype had significantly higher lactose content than those with the GG genotype, suggesting potential use as a molecular genetic marker for dairy cow breeding. A method for assessing lactose content in cow milk has been developed. This method is simple, reliable, and significantly reduces costs compared to high-throughput sequencing. This method can be used for molecular breeding of dairy cows for production performance in my country, thereby enabling the establishment of a dairy herd with superior genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Figure 1 The figure is the electrophoresis diagram of PCR amplification of the LBP target fragment; M is DL2,000 DNA Marker, and lanes 1 and 2 are the electrophoresis results of amplifying the LBP target fragment using the DNA pool as a template.
[0043] Figure 2 This is the sequencing result of the pooled PCR amplified LBP target fragment.
[0044] Figure 3 The results of amplifying the LBP target fragment using a single sample DNA as a template; M is DL2,000 DNA Marker, and lanes 1 to 8 are the results of amplifying the target fragment using individual DNA.
[0045] Figure 4 The results of enzyme digestion of PCR products; M is DL2,000 DNA Marker, and the individual genotypes in lanes 1 to 5 are GG, AA, GA, GA, and AA, respectively. DETAILED DESCRIPTION
[0046] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.
[0047] Example 1 Design of primers for bovine LBP gene sequence amplification and sequence amplification
[0048] 1. Experimental Methods
[0049] 1. Sample acquisition and preparation of DNA pools
[0050] A total of 203 Holstein cows housed in the same environment were selected. Blood was collected from the jugular vein and anticoagulated. The collected blood samples were stored at -80°C. DNA from individual samples was extracted using a blood DNA extraction kit (Beijing Tiangen) to obtain individual whole-genome DNA and DNA pools, which were then stored at -20°C.
[0051] 2. Design and synthesis of primers for amplification of bovine LBP gene sequence
[0052] According to the bovine LBP gene sequence published in Ensembl (ENSBTAT00000022428.3), primers were designed: upstream primer sequence: 5'-TGAAGGCTTGACAACGGAG-3' (SEQ ID NO: 1); downstream primer sequence: 5'-AGACCCACAGAAGAGGCATC-3' (SEQ ID NO: 2). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0053] 3. DNA pool PCR amplification and sequencing
[0054] PCR amplification was performed using a pool of Holstein cow DNA as a template. The PCR system is shown in Table 1. The PCR amplification procedure is shown in Table 2. PCR products were detected by 1.5% agarose gel electrophoresis at 120 V, 400 mA, and 15 min. After confirming the presence of a single band of the correct length and the absence of specific bands, the product was sequenced by Sangon Biotech (Shanghai) Co., Ltd.
[0055] 4. Results Analysis
[0056] Overlapping peaks were observed using Chromas software.
[0057] Table 1. PCR reaction system
[0058]
[0059]
[0060] Table 2. PCR reaction program
[0061]
[0062] 2. Experimental Results
[0063] The gene sequence amplified by the primers is 624 bp in length. Figure 1 The results showed a single, clear PCR product band with good specificity, no smearing, and no nonspecific bands. The band lengths were consistent with the target band, as indicated by the DL2,000 DNA Marker.
[0064] The sequencing results of PCR amplification products are as follows Figure 2An overlapping peak was found at 237 bp of the amplified product. Mapping the mutation to the LBP gene sequence revealed a mutation at nucleotide position 67219155 on chromosome 13 of the LBP genome, where the mutation occurred from G to A (i.e., I2-1095 G>A).
[0065] Example 2 Amplification of individual dairy cow LBP gene sequence and PCR-RFLP identification
[0066] 1. Experimental Methods
[0067] 1. Amplification of LBP gene sequence of individual dairy cows to be tested
[0068] Amplification was performed using the whole genomic DNA from the blood of the 203 Holstein cows in Example 1 as a template. The amplification system and reaction procedure were the same as in Example 1. PCR products were detected by 1.5% agarose gel electrophoresis under the following conditions: 120 V, 400 mA, 15 min.
[0069] 2. PCR-RFLP genotyping
[0070] PCR products were digested with the restriction endonuclease Afl II (see Table 3 for the enzyme digestion system). The reaction system was incubated at 37°C for 2 hours. 5 μl of the digested product was subjected to 2.5% agarose gel electrophoresis to determine the band length for genotyping. Electrophoresis conditions: 80 V, 400 mA, 10 min.
[0071] Table 3. Enzyme digestion system
[0072]
[0073] 2. Experimental Results
[0074] The results of LBP gene amplification electrophoresis of individual dairy cows are as follows Figure 3 Electrophoresis results showed a single, clear PCR product band with good specificity, and no smearing or nonspecific bands. Using the DL2,000 DNA Marker, the band length matched the target band, confirming the conditions for subsequent enzyme digestion experiments.
[0075] The results of restriction endonuclease Afl II digestion are as follows Figure 4, the restriction enzyme site is located at the 234th bp of the PCR product. If the base at the SNP site is G, it shows a single band of 624 bp; if the base is A, it shows two bands of 234 bp and 390 bp. Correspondingly, the electrophoresis results of GG genotype individuals show a single band of 624 bp; the electrophoresis results of GA genotype individuals show three bands of 624 bp, 234 bp and 390 bp; the electrophoresis results of AA genotype individuals show two bands of 234 bp and 390 bp.
[0076] Example 3 Correlation analysis between the I2-1095 G>A locus of the dairy cow LBP gene and dairy cow milk quality traits
[0077] I. Experimental method
[0078] According to the results of Example 2, use Popgen32 to count the gene frequencies, genotype frequencies, effective number of alleles (Ne), and genetic heterozygosity (H) of 203 Holstein dairy cows in Example 1; use PIC software to calculate the polymorphism information content (PIC); use SPSS 23.0 software to perform a significant difference test on the I2-1095 G>A locus and milk quality traits. P<0.05 indicates a significant difference, P<0.01 indicates a highly significant difference, and the trait values are expressed as "average ± standard error".
[0079] II. Experimental results
[0080] The gene frequencies of alleles G and A at the I2-1095 G>A locus are 0.6814 and 0.3186 respectively, and the dominant allele is G. There are three genotypes at this locus, namely GG, GA, and AA, and their genotype frequencies are 0.4706, 0.4216, and 0.1078 respectively, and the dominant genotype is GA. The chi-square value is 0.173, P>0.05, indicating that this locus conforms to the Hardy-Weinberg equilibrium state in the population.
[0081] [[ID=第十八]]The polymorphism information content (PIC) is used to detect the distribution of allele frequencies and evaluate the polymorphism of alleles in the population. When PIC<0.25, it indicates that the alleles are in low polymorphism in the population; when 0.25<PIC<0.50, it indicates that the alleles are in moderate polymorphism in the population; PIC>0.50 indicates that the alleles are in high polymorphism in the population. The PIC value of the two alleles at this SNP locus is 0.34, and the genetic heterozygosity (H) value is 0.4342, both of which are between 0.25 and 0.50, indicating that alleles G and A are in moderate polymorphism in this population.
[0082] Table 4. Population genetic parameters of the I2-1095 G>A locus of the LBP gene
[0083]
[0084] Note: 2 The value did not reach the significant level (P>0.05), χ 2 0.05 (df=2)=5.99
[0085] Example 4 Correlation between the genotype of the I2-1095 G>A locus of the LBP gene in dairy cows and milk quality traits.
[0086] 1. Experimental Methods
[0087] The eight indicators of milk quality traits of the 203 Holstein cows in Example 1, including milk production, milk fat, milk protein, lactose, dry matter, somatic cells, urea nitrogen, and corrected milk, were tested, and data statistics were performed, and the association between different genotypes of the LBP gene SNP site and milk quality traits was analyzed.
[0088] 2. Experimental Results
[0089] Among the 203 dairy cow samples tested, the average data of eight indicators including milk production, milk fat, milk protein, lactose, dry matter, somatic cells, urea nitrogen, and corrected milk for the three genotypes at the I2-1095 G>A locus are shown in Table 5.
[0090] Table 5. Correlation analysis between LBP gene polymorphism and milk quality traits in dairy cows
[0091]
[0092] Note: No lowercase letters indicate extremely significant differences (P<0.01), and no lowercase letters indicate non-significant differences (P>0.05)
[0093] Association analysis showed that, except for lactose, the seven indicators were not significantly associated with the three genotypes at the I2-1095 G>A locus (P>0.05). However, the different genotypes at the SNP locus were extremely significantly associated with the lactose trait. Individuals with the GA genotype at the I2-1095 G>A locus of the LBP gene had a significantly higher lactose trait than those with the GG genotype (P<0.01).
[0094] Example 5 A method for evaluating the lactose content in cow milk
[0095] 1. Extract DNA from individual samples
[0096] 2. PCR Amplification and Sequencing
[0097] PCR amplification was performed using a pool of Holstein cow DNA as a template.
[0098] Upstream primer sequence: 5'-TGAAGGCTTGACAACGGAG-3' (SEQ ID NO: 1),
[0099] Downstream primer sequence: 5′-AGACCCACAGAAGAGGCATC-3′ (SEQ ID NO: 2);
[0100] The PCR system was as follows: Green Taq Mix 10 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, total volume 20 μL.
[0101] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.
[0102] PCR products were detected by 1.5% agarose gel electrophoresis at 120 V, 400 mA, and 15 min. After confirming that the bands were single, of the correct length, and without specific bands, sequencing was performed.
[0103] 3. Interpretation of results
[0104] There is a SNP site located at ARS-UCD1.2 version NC_037340.1:67219155, that is, nucleotide 67219155 on chromosome 13 of the LBP genome. The lactose content in the milk of individual cows with the genotype GA is higher than that in the milk of individual cows with the genotype GG.
[0105] Example 6 A kit for evaluating the lactose content in cow milk
[0106] 1. Composition
[0107] The nucleotide sequences are shown in SEQ ID NO: 1-2, the primers and PCR reagents (Green Taq Mix and ddH2O).
[0108] 2. Usage
[0109] As in Example 5.
[0110] Example 7 A method for evaluating the lactose content in cow milk
[0111] 1. Extract DNA from individual samples
[0112] 2. PCR Amplification and Sequencing
[0113] PCR amplification was performed using a pool of Holstein cow DNA as a template.
[0114] Upstream primer sequence: 5'-TGAAGGCTTGACAACGGAG-3' (SEQ ID NO: 1),
[0115] Downstream primer sequence: 5′-AGACCCACAGAAGAGGCATC-3′ (SEQ ID NO: 2);
[0116] The PCR system was as follows: Green Taq Mix 10 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DNA template 1.0 μL, ddH2O 8 μL, total volume 20 μL.
[0117] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 45 s, 72°C for 1 min, 30 cycles; 72°C for 5 min.
[0118] PCR products were examined by 1.5% agarose gel electrophoresis at 120 V, 400 mA, and 15 min to confirm that the bands were single, of the correct length, and free of specific bands.
[0119] 3. PCR-RFLP genotyping
[0120] The PCR product was digested with restriction endonuclease Afl II. The digestion system was as follows: 14 μL of PCR product, 0.2 μL of Afl II enzyme, 2 μL of 10× rCutSmart Buffer, and 3.8 μL of ddH2O, with a total volume of 10 μL.
[0121] The reaction system was incubated in a 37°C incubator for 2 h. 5 μl of the digested product was subjected to 2.5% agarose gel electrophoresis to detect the band length for genotyping. Electrophoresis conditions: 80 V, 400 mA, 10 min.
[0122] IV. Interpretation of results
[0123] There is a SNP site located at ARS-UCD1.2 version NC_037340.1:67219155, that is, nucleotide 67219155 on chromosome 13 of the LBP genome. The lactose content in the milk of individual cows with the genotype GA is higher than that in the milk of individual cows with the genotype GG.
[0124] After enzyme digestion, the individuals with one band of 624bp in electrophoresis were GG genotype individuals, the individuals with three bands of 624bp, 234bp and 390bp were GA genotype individuals, and the individuals with two bands of 234bp and 390bp were AA genotype individuals.
[0125] Example 8 A kit for evaluating the lactose content in cow milk
[0126] 1. Composition
[0127] The nucleotide sequences are shown in SEQ ID NOs: 1 to 2, the primers, PCR reagents (Green Taq Mix and ddH2O), and enzyme digestion reagents (Afl II enzyme, 10× rCutSmart Buffer, and ddH2O).
[0128] 2. Usage
[0129] As in Example 7. Sequence Listing <110> Guangdong Ocean University <120> A method for evaluating lactose content in cow milk <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 tgaaggcttg acaacggag 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 agacccacag aagaggcatc 20
Claims
1. Use of a reagent for detecting the genotype of ARS-UCD version 1.2 NC_037340.1: 67219155 in evaluating the lactose content in the milk of Holstein cows or preparing a kit for evaluating the lactose content in the milk of Holstein cows, characterized in that: The lactose content in the milk of individual cows with genotype GA is higher than that of individual cows with genotype GG.
2. A method for evaluating the lactose content in Holstein cow milk, characterized in that: The genotype of ARS-UCD1.2 version NC_037340.1:67219155 was detected. The lactose content in the milk of individual cows with the genotype GA was higher than that of individual cows with the genotype GG.
3. The method according to claim 2, characterized in that The genotype of ARS-UCD1.2 version NC_037340.1:67219155 was detected using primers with nucleotide sequences as shown in SEQ ID NOs: 1-2. The lactose content in the milk of individual cows with the genotype GA was higher than that in the milk of individual cows with the genotype GG.
4. The method according to claim 3, characterized in that The PCR product of the primers was digested with endonuclease Afl II. After digestion, the individuals with one band of 624 bp on electrophoresis were GG genotype individuals, those with three bands of 624 bp, 234 bp and 390 bp were GA genotype individuals, and those with two bands of 234 bp and 390 bp were AA genotype individuals.
5. Use of a reagent for evaluating the lactose content in Holstein cow milk in molecular breeding for the lactose content in Holstein cow milk, characterized in that: The reagent is used to detect the genotype of ARS-UCD1.2 version NC_037340.1: 67219155, and the lactose content in the milk of individual cows with the genotype GA is higher than the lactose content in the milk of individual cows with the genotype GG.
6. Application of the method according to claim 3 in molecular breeding for lactose content in Holstein cow milk.
7. Use of a kit for evaluating lactose content in Holstein cow milk in molecular breeding for lactose content in Holstein cow milk, characterized in that: The kit contains a reagent for detecting the genotype of ARS-UCD1.2 version NC_037340.1: 67219155, wherein the lactose content in the milk of individual cows with the genotype GA is higher than that in the milk of individual cows with the genotype GG.
8. The use according to claim 7, characterized in that The reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.
9. The use according to claim 8, characterized in that Also contains PCR reagents.
10. The use according to claim 8 or 9, characterized in that: Also contains endonuclease Afl II and 10× CutSmart Buffer.