A method for evaluating lactose content in milk quality traits of chinese holstein cows

Abstract

The application discloses a method for evaluating lactose content in milk quality traits of Chinese Holstein cows, detects a site of rs109894153 or 4:g.27093247C>T (ENSBTAT00000072753.1:c.-439C>T) at position 27093247 of chromosome 4 in ARS-UCD version 1.2 bovine genome, and genotypes of the SNP site, wherein the lactose content of milk of an individual with TT genotype is higher than that of an individual with CT genotype. Meanwhile, a kit for evaluating lactose content in milk quality traits of Chinese Holstein cows is established by using the SNP site. The SNP site disclosed by the application can play an important role in molecular breeding of lactose content in milk quality traits of Chinese Holstein cows, and can be used for producing dairy products suitable for different lactose requirements.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and more specifically, to a method for evaluating lactose content in milk quality traits of Chinese Holstein dairy cows. Background Technology

[0002] Holstein cattle are even-toed ungulates belonging to the family Bovidae and genus Bos, and are currently the world's most productive and widely raised dairy cattle breed. Due to their high milk production, they were introduced to my country in the late 19th century and crossbred with Chinese yellow cattle. Through long-term selective breeding, the Chinese Holstein cattle were developed.

[0003] Lactose is a carbohydrate unique to human and mammalian milk. It is a disaccharide composed of glucose and galactose, with the molecular formula C6H2O. 12 H 22 O 11 Lactose is hydrolyzed into glucose and galactose by lactase secreted by the brush border of the small intestinal epithelial cells. Glucose primarily provides energy for the body, while galactose binds to ceramides via glycosidic bonds, forming galactocerebroside, which participates in brain development. During the growth and development of infants and young children, lactose not only provides energy but also participates in the process of brain development.

[0004] Meanwhile, some people experience digestive symptoms such as diarrhea, bloating, and abdominal pain after consuming dairy products. This is because the activity of lactase is reduced, lacking, or even absent, leading to poor absorption of lactose in the intestines. Undigested lactose enters the colon and is fermented by bacteria to produce gases (hydrogen, carbon dioxide, etc.) and short-chain fatty acids. The large amount of gas causes symptoms such as bloating, while the undigested lactose and its fermentation products increase the osmotic pressure in the intestines, increasing the amount of water flowing into the intestines and causing diarrhea. This condition is called lactose intolerance.

[0005] Therefore, the dairy market demands dairy products with different lactose contents, making the breeding of Chinese Holstein cows that secrete different lactose contents of great economic value and significance.

[0006] Histone deacetylase 9 (HDAC9) is a member of family IIa of histone deacetylases. HDAC9 can freely shuttle between the nucleus and cytoplasm, promoting tissue-specific transcriptional regulation through interactions with histone and non-histone substrates. HDAC9 plays a crucial role in various physiological processes, including cardiac development, bone formation, adipocyte differentiation, and innate immunity. Currently, numerous biological studies on HDAC9 have been reported and progress has been made, primarily focusing on humans and mice: HDAC9 silencing inhibits oxygen-glucose deprivation / reperfusion injury in neonatal rat hippocampal neurons; altered HDAC9 expression levels in mouse brain tissue after cerebral infarction; the regulatory role of histone deacetylase 9 in glomerular development; and the HDAC9-MALAT1-BRG1 complex mediating smooth muscle dysfunction in thoracic aortic aneurysms. No correlation was found between HDAC9 and lactose content in dairy products. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for evaluating the lactose content in the milk quality traits of Chinese Holstein cows.

[0008] The first objective of this invention is to provide a method for evaluating the lactose content in milk quality traits of Chinese Holstein cows.

[0009] A second objective of this invention is to provide a kit for evaluating lactose content in milk quality traits of Chinese Holstein cows.

[0010] A third objective of this invention is to provide a reagent for detecting the genotype of SNP loci in the application of assessing lactose content in milk quality traits of Chinese Holstein dairy cows.

[0011] A fourth objective of this invention is to provide the application of reagents for detecting the genotype of SNP loci in the preparation of a kit for assessing lactose content in milk quality traits of Chinese Holstein cows.

[0012] A fifth objective of this invention is to provide the application of the reagents, any of the methods described herein, and / or any of the kits described herein for detecting the genotype of SNP loci in molecular breeding of lactose content in milk quality traits of Chinese Holstein dairy cows.

[0013] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0014] This invention discovered a polymorphic site in the HDAC9 gene through direct sequencing, analyzed its genetic effects in the population, and then conducted an association analysis with the milk quality traits of selected Holstein cows, finding that its genotype was significantly correlated with lactose content in milk quality traits.

[0015] Therefore, this invention requires a method for assessing lactose content in milk quality traits of Chinese Holstein dairy cows, by detecting the genotype of the SNP locus at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, namely the rs109894153 locus.

[0016] Preferably, primers with nucleotides such as those shown in SEQ ID NO:1-2 are used to amplify the DNA of the individual to be tested.

[0017] Upstream sequence: 5'-AAGGGAGGAAAGAGTTAATGGC-3' (SEQ ID NO:1);

[0018] The downstream sequence 5'-ACGGCTCAGTGATGAAGAAAG-3' (SEQ ID NO:2)

[0019] Preferably, at the SNP locus, the lactose content in the milk of individuals with the TT genotype is higher than that of individuals with the CT genotype.

[0020] As a specific example, the PCR reaction adjustment program is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 min, cycled 35 times; extension at 72℃ for 10 min, and stored at 16℃ for later use.

[0021] PCR reaction system (20 μL): 10 μL Taq PCR Mix, including 0.5 μL each of upstream and downstream primers, 2 μL of selected template DNA, and 7 μL of deionized water.

[0022] And, a kit for assessing lactose content in milk quality traits of Chinese Holstein cows, containing a reagent for detecting the genotype of the SNP locus at position 27093247 on chromosome 4 of the ARS-UCD version 1.2 bovine genome, namely the rs109894153 locus.

[0023] Preferably, the reagent is a primer of nucleotides as shown in SEQ ID NO:1-2.

[0024] Preferably, it also contains PCR amplification reagents.

[0025] Preferably, the PCR amplification reagent is Taq PCR Mix.

[0026] It also requires protection of the use of reagents for detecting the genotype of SNP loci in assessing lactose content in milk quality traits of Chinese Holstein dairy cows, wherein the SNP locus is located at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, namely rs109894153.

[0027] And the application of reagents for detecting the genotype of SNP sites in the preparation of a kit for assessing lactose content in milk quality traits of Chinese Holstein dairy cows, wherein the SNP site is located at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, i.e., rs109894153.

[0028] The application of the reagents for detecting the genotype of SNP loci, any of the methods described herein, and / or any of the kits described herein in the molecular breeding of lactose content in milk quality traits of Chinese Holstein dairy cows is also within the scope of protection of this invention.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention discloses a method for assessing lactose content in the milk quality traits of Chinese Holstein dairy cows. The method detects the genotype of a SNP locus located at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, specifically the rs109894153 locus or the 4:g.27093247C>T(ENSBTAT00000072753.1:c.-439C>T). Individuals with the TT genotype have higher lactose content in their milk than those with the CT genotype. A kit for assessing lactose content in the milk quality traits of Chinese Holstein dairy cows is also developed using this SNP locus. The SNP locus of this invention can play an important role in molecular breeding for lactose content in Chinese Holstein dairy cows, enabling the production of dairy products adapted to different lactose requirements. Attached Figure Description

[0031] Figure 1 The results are PCR amplification of mixed pool DNA.

[0032] Figure 2 Sequencing results for three genotypes at the HDAC9 gene locus 4:g.27093247C>T(rs109894153). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0034] Example 1

[0035] I. Experimental Methods

[0036] 1. Experimental Samples

[0037] 112 Chinese Holstein dairy cows were raised under the same conditions in the same location. Venous blood (5 mL / cow) was collected from the same parts of the cows and then treated with anticoagulants before being stored at low temperature (-80℃) for later use.

[0038] 2. DNA extraction

[0039] A blood genomic DNA extraction kit was used to extract DNA from blood samples collected from 112 Chinese Holstein dairy cows, following the instructions in the kit. After extraction, the purity and concentration of the extracted DNA were tested using a Nano-Drop ND-2000 microspectrophotometer, and the quality of the DNA was determined by agarose gel electrophoresis.

[0040] 3. Primer design

[0041] Based on the bovine HDAC9 gene sequence (ENSBTAT00000072753.1) published in Ensembl, primers were designed...

[0042] Upstream sequence: 5'-AAGGGAGGAAAGAGTTAATGGC-3' (SEQ ID NO:1);

[0043] The downstream sequence 5'-ACGGCTCAGTGATGAAGAAAG-3' (SEQ ID NO:2)

[0044] It was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0045] 4. PCR amplification and product detection

[0046] Genomic DNA from 112 Chinese Holstein dairy cows was mixed as pooled DNA and amplified by PCR. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min, and storage at 16℃ for later use.

[0047] PCR reaction system (20 μL): 10 μL Taq PCR Mix. This includes 0.5 μL each of upstream and downstream primers, 2 μL of selected template DNA, and 7 μL of deionized water.

[0048] After the PCR products were detected by 1.5% agarose gel electrophoresis, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0049] II. Experimental Results

[0050] PCR amplification results of mixed pool DNA are as follows Figure 1 The results showed that the primer amplification had good specificity. The PCR product size was 869 bp, and the result indicated that the PCR product size was consistent with the expected fragment size, which could be directly used for sequencing identification. The expected fragment was SEQ ID NO: 3, i.e.:

[0051] .

[0052] The results showed that the HDAC9 gene has a SNP site located at 297 bp of the target fragment (SEQ ID NO:3). This site has three genotypes, namely TT, CC and CT, which are located at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, namely rs109894153 or 4:g.27093247C>T(ENSBTAT00000072753.1:c.-439C>T).

[0053] Example 2: Genetic characteristics of the HDAC9 gene 4:g.27093247C>T site in a population. I. Experimental methods

[0054] Following the method in Example 1, the DNA of 112 Chinese Holstein dairy cows from Example 1 was amplified by PCR and sequenced. The sequencing results were analyzed using SeqMan to obtain the genotype of each Chinese Holstein dairy cow at the 4:g.27093247C>T locus.

[0055] Then, Popgene software was used to calculate the gene frequency, genotype frequency, heterozygosity (H), effective allele count (Ne), and polymorphism information content (PIC) of its alleles, and to calculate X. 2 value.

[0056] II. Experimental Results

[0057] Figure 2 Sequencing results for three genotypes at the HDAC9 gene locus 4:g.27093247C>T. The population genetic characteristics of the 4:g.27093247C>T locus are shown in Table 1. The results show that the dominant genotype at this locus is TT, the dominant allele is T, and its frequency is greater than 0.5. The heterozygosity (H) and polymorphism information content (PIC) of the 4:g.27093247C>T locus are both between 0.25 and 0.50, indicating that this locus exhibits moderate polymorphism in the HDAC9 gene. 2 Test result 4: The g.27093247C>T site is in Hardy-Weinberg equilibrium in the Chinese Holstein dairy cattle population (P>0.05).

[0058]

[0059] Note: χ 2 The superscript indicates that the value was not significant (P>0.05).

[0060] Example 3: Correlation between the HDAC9 gene 4:g.27093247C>T site and milk quality traits in Chinese Holstein dairy cows

[0061] I. Experimental Methods

[0062] The milk quality traits of the milk produced by the 112 Chinese Holstein dairy cows in Example 1 were tested using the following specific methods:

[0063] Six indicators were measured in individual Chinese Holstein cattle: milk yield (kg), milk fat content (%), milk protein content (%), lactose content (%), dry matter content (%), and urea nitrogen content (mg / L). The specific testing method was as follows: The milk quality traits of 112 dairy cows in Example 1, which were raised in the same environment and were in lactation, were tested using a milk composition analyzer. These traits included milk fat content (%), milk protein content (%), lactose content (%), urea nitrogen content (mg / L), and dry matter content (%).

[0064] Individual milk production, which is the milk production of each dairy cow at each lactation stage, is the basis for milk production statistics. Individual milk production refers to the milk production from the first day after calving to the 305th day.

[0065] Next, a one-way ANOVA with multiple comparisons was used in SPSS 23.0 software to test the significance of differences in the relationship between milk production traits and genotypes of the selected dairy cows (P < 0.05 indicates significant difference, and P < 0.01 indicates highly significant difference). The final results are expressed as mean ± standard deviation.

[0066] II. Experimental Results

[0067] The correlation analysis results between the HDAC9 gene 4:g.27093247C>T site and milk quality traits in Chinese Holstein dairy cows are shown in Table 2. Among the milk quality traits associated with this SNP site, lactose showed a significant correlation (P<0.05), with the TT genotype exhibiting higher lactose levels. No significant correlations were found between milk fat, milk yield, milk protein, urea nitrogen, and dry matter content (P<0.05).

[0068] Table 2

[0069]

[0070] Example 4: A method for evaluating milk quality traits of Chinese Holstein dairy cows

[0071] I. Extraction of DNA from Chinese Holstein Dairy Cows

[0072] II. PCR Amplification and Product Detection

[0073] Chinese Holstein dairy cow DNA was used as a template for PCR amplification. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min, and storage at 16℃ for later use.

[0074] PCR reaction system (20 μL): 10 μL Taq PCR Mix, 0.5 μL each of upstream and downstream primers, 2 μL of selected template DNA, and 7 μL deionized water.

[0075] III. Result Interpretation

[0076] 4:g.27093247C>T, located at 297bp of the amplification product (SEQ ID NO:3), is located at position 27093247 on chromosome 4 of the bovine genome in ARS-UCD version 1.2, i.e., rs109894153 or 4:g.27093247C>T(ENSBTAT00000072753.1:c.-439C>T); three genotypes are present: TT, TC, and CC; the lactose content of milk from individuals with the TT genotype is higher than that from individuals with the CT genotype.

[0077] Example 5: A kit for evaluating milk quality traits of Chinese Holstein cows

[0078] I. Composition

[0079] Nucleotides as shown in SEQ ID NO:1-2, primers, Taq PCR Mix, and deionized water.

[0080] II. Instructions for Use

[0081] Same as Example 4.

Claims

1. A method for evaluating lactose content in milk quality traits of Chinese Holstein dairy cows, characterized in that, The genotype of SNP locus rs109894153 was detected. Individuals with the TT genotype at this SNP locus had higher lactose content in their milk than individuals with the CT genotype.

2. The method according to claim 1, characterized in that, The DNA of the individual to be tested was amplified using primers with nucleotide sequences as shown in SEQ ID NO:1-2.

3. A kit for assessing lactose content in milk quality traits of Chinese Holstein dairy cows, and its application in molecular breeding of lactose content in milk quality traits of Chinese Holstein dairy cows, characterized in that... The kit contains reagents for detecting the genotype of the SNP locus rs109894153, where individuals with the TT genotype at the SNP locus have higher lactose content in their milk than individuals with the CT genotype.

4. The application according to claim 3, characterized in that, The reagent is a primer with a nucleotide sequence as shown in SEQ ID NO:1-2.

5. The application according to claim 3 or 4, characterized in that, The kit also contains PCR amplification reagents.

6. The application according to claim 5, characterized in that, The PCR amplification reagent is Taq PCR Mix.

7. The application of a reagent for detecting the genotype of SNP locus rs109894153 in assessing lactose content in milk quality traits of Chinese Holstein dairy cows, characterized in that... Individuals with the TT genotype at the SNP locus had higher lactose content in their milk than individuals with the CT genotype.

8. The application of a reagent for detecting the genotype of SNP locus rs109894153 in the preparation of a kit for assessing lactose content in milk quality traits of Chinese Holstein dairy cows, characterized in that... Individuals with the TT genotype at the SNP locus had higher lactose content in their milk than individuals with the CT genotype.

9. The application of the method according to any one of claims 1 to 2 in molecular breeding for lactose content in milk quality traits of Chinese Holstein dairy cows.

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