Method for evaluating corrected milk yield of Chinese Holstein dairy cow

Abstract

The invention discloses a method for evaluating corrected milk yield of Chinese Holstein cows, the genotype of rs109669792 is detected, and the corrected milk yield of a GG genotype individual is obviously higher than that of a GA type individual. According to the invention, SNP--rs109669792 which is obviously related to the corrected milk yield is found in Chinese Holstein cows, and the corrected milk yield of a GG genotype individual is obviously higher than that of a GA type individual. Furthermore, a primer for detecting the genotype of the SNP locus is utilized for detecting the genotype of the SNP locus, so that the corrected milk yield of the Chinese Holstein dairy cow can be evaluated, and the primer can be used for molecular breeding in the aspect of the milk quality character of the Chinese Holstein dairy cow and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of breeding of Chinese Holstein dairy cows, and in particular to a method for evaluating the corrected milk yield of Chinese Holstein dairy cows. Background Art

[0002] Chinese Holstein cows, as an outstanding result of the cross between European Holstein cows and Chinese yellow cattle, have become a unique dairy cow breed in my country. They not only have a large milk production, a large number, and a wide distribution, but also can be used for both milk and meat. In addition, this breed is tall, gentle, and easy to manage. With the improvement of the national economic level and the enhancement of people's health awareness, milk consumption has gradually increased. Therefore, it is necessary to continuously select and breed excellent dairy cow breeds and improve the quality of dairy products to meet market demand.

[0003] Kinesin family binding protein (KIFBP) has 621 amino acids, including 2 tetrapeptide repeats, and is expressed in the central nervous system and enteric nervous system. It binds to eight kinesins in cells, including members of the kinesin-2 (KIF3A), kinesin-3 (KIF1A, KIF1B, KIF1C, KIF13B, and KIF14), kinesin-8 (KIF18A), and kinesin-12 (KIF15) families, thereby inhibiting motor-microtubule binding in vitro and in cells. Kinesin binding protein interacts with multiple kinesins, regulates protein activity by binding to kinesins, and blocks the interaction between kinesins and the microtubule skeleton; it is also involved in the structure and growth of axons, microtubule dynamics, and cargo transport, and exerts its effects by binding to various microtubule-associated proteins. Studies have shown that knockout of the KIFBP gene in mice results in defects in the central and peripheral nervous systems and delayed development of the enteric nervous system; knockout of the zebrafish KIFBP ortholog leads to disruption of axon structure and growth, including axon defects in the enteric nervous system. Overexpression of KIFBP inhibits the function of the motor protein, thereby blocking the intracellular transport of the virus and inhibiting the proliferation of the virus.

[0004] In addition, the KIFBP gene is also the causative gene for Goldberg-Shprintzen syndrome (GOSHS), an autosomal recessive neurological disorder characterized by defects in neuronal development, mental retardation, facial dysmorphism, heart defects, and often Hirschsprung's disease. To date, 16 KIFBP mutations that cause GOSHS have been discovered; most mutations result in reduced or complete loss of protein expression. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a method for evaluating the corrected milk yield of Chinese Holstein cows.

[0006] The first object of the present invention is to provide a method for evaluating the corrected milk yield of Chinese Holstein cows.

[0007] The second object of the present invention is to provide a primer for evaluating the corrected milk yield of Chinese Holstein cows.

[0008] The third object of the present invention is to provide a reagent for detecting the genotype of rs109669792 and its application in evaluating the corrected milk yield of Chinese Holstein cows.

[0009] The fourth object of the present invention is to provide a use of a reagent for detecting the genotype of rs109669792 in preparing a kit for evaluating the corrected milk yield of Chinese Holstein cows.

[0010] A fifth object of the present invention is to provide a kit for evaluating the corrected milk yield of Chinese Holstein cows.

[0011] The sixth object of the present invention is to provide a method for breeding Chinese Holstein cows with high corrected milk yield.

[0012] The seventh object of the present invention is to provide the method, the primer or the application of the method in molecular breeding of Chinese Holstein cows with high corrected milk yield.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] A method for evaluating the corrected milk yield of Chinese Holstein cows, comprising detecting the genotype of rs109669792; the rs109669792 locus is located at NC_037355.1:25247823, i.e., position 25247823 of chromosome 28 of the ARS-UCD 1.3 version of the bovine genome, and has three genotypes: AA, AG and GG, wherein the corrected milk yield of individuals with the GG genotype is significantly higher than that of individuals with the GA genotype.

[0015] Preferably, the genotype of rs109669792 is detected by PCR amplification of the sample DNA using primers shown in the nucleotide sequences SEQ ID NOs: 1-2.

[0016] More preferably, the reaction system of the PCR amplification is: 1 μL of template DNA, 12.5 μL of Taq PCR Mix, 0.7 μL of each primer, and 10.1 μL of deionized water.

[0017] More preferably, the reaction procedure of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 40 cycles; 72°C for 5 min.

[0018] The present invention also claims a primer for evaluating the corrected milk yield of Chinese Holstein cows, characterized in that the nucleotide sequence of the primer is shown in SEQ ID NO: 1-2.

[0019] The upstream primer sequence was 5′-TCCACTGATGGGCTGGGTTA-3′ (SEQ ID NO: 1);

[0020] The downstream primer sequence was 5′-TGGTCTCGGCAAGAGTTGAG-3′ (SEQ ID NO: 2).

[0021] The following applications are also claimed:

[0022] Application of reagents for detecting the genotype of rs109669792 in evaluating the corrected milk yield of Chinese Holstein cows.

[0023] Application of a reagent for detecting the genotype of rs109669792 in preparing a kit for evaluating the corrected milk yield of Chinese Holstein cows.

[0024] The present invention also claims a kit for evaluating the corrected milk volume of Chinese Holstein cows, comprising a reagent for detecting the genotype of rs109669792.

[0025] Preferably, the reagent is a primer shown in the nucleotide sequence SEQ ID NO: 1-2.

[0026] The present invention also claims a method for breeding Chinese Holstein cows with high corrected milk yield, which is obtained by using the method described in claim 1 to select Chinese Holstein cows with high corrected milk yield as parents for breeding.

[0027] The method, the primers or the application of the method in molecular breeding of Chinese Holstein cows with high corrected milk yield also fall within the protection scope of the present invention.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention discovered a SNP—rs109669792—significantly associated with corrected milk yield in Chinese Holstein cows, and the corrected milk yield of individuals with the GG genotype was significantly higher than that of individuals with the GA genotype. Furthermore, primers for detecting the genotype of the SNP site can be used to evaluate the corrected milk yield of Chinese Holstein cows, and can be used for molecular breeding of corrected milk yield in Chinese Holstein cows, which has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the electrophoresis of PCR products using Chinese Holstein cow pooled DNA as template; M: DL2000 DNA Marker; 1: KIFBP gene PCR amplification product.

[0031] Figure 2 These are the sequencing results of three genotypes at the rs109669792G>A site of the KIFBP gene. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example 1 PCR amplification of the KIFBP gene of Chinese Holstein cows

[0034] 1. Experimental Methods

[0035] 1. Sample preparation

[0036] 92 Chinese Holstein dairy cows were raised under the same environment and conditions in a cattle farm in Heilongjiang Province. 5 mL of blood was collected from the jugular vein of each cow, anticoagulated with sodium citrate, and stored at 4°C.

[0037] The DNA of the above samples was extracted using a DNA extraction kit and stored in an environment of -20°C. 40 to 50 different DNA samples were randomly selected to ensure that there was no duplication between them, and a DNA mixed pool sample was prepared.

[0038] 2. Primer design

[0039] Based on the bovine KIFBP gene sequence published in the Ensembl database (ENSBTAT00000019353.4), specific primers were designed, wherein the upstream primer sequence was 5′-TCCACTGATGGGCTGGGTTA-3′ (SEQ ID NO: 1); the downstream primer sequence was 5′-TGGTCTCGGCAAGAGTTGAG-3′ (SEQ ID NO: 2).

[0040] 3. PCR amplification and electrophoresis detection

[0041] The DNA pool samples were amplified by PCR using the primers shown in SEQ ID NOs: 1-2.

[0042] The reaction system for PCR amplification was 25 μL: 1 μL template DNA, 12.5 μL Taq PCR Mix, 0.7 μL each primer, and 10.1 μL deionized water.

[0043] The reaction procedure of PCR amplification was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 5 min, and storage at 4°C for future use.

[0044] The PCR product was detected by 1.5% agarose gel electrophoresis and the target band was confirmed to be correct, and then sent to Qingke Biotechnology (Guangzhou) Co., Ltd. for sequencing.

[0045] 2. Experimental Results

[0046] PCR amplification was performed using the primers shown in SEQ ID NO: 1-2 as templates for mixed pool DNA. Figure 1 As shown, the amplified product is a 903 bp fragment, and the electrophoresis pattern is clear, without primer dimers, fuzzy bands and non-specific amplification. It can be directly used for sequencing identification, and the product sequence is SEQ ID NO: 3, that is:

[0047]

[0048] (The primers shown in SEQ ID NO: 1-2 are underlined, and the site marked in grey is rs109669792).

[0049] Sequencing showed that there was an obvious overlapping peak, indicating that there was a SNP mutation site in the PCR product, namely rs109669792, located at NC_037355.1:25247823, that is, position 25247823 of chromosome 28 of the ARS-UCD 1.3 version of the bovine genome. There were three genotypes: AA, AG and GG. The relevant genotype peak results are shown in Figure 2 .

[0050] Example 2 Population genetic characteristics of the KIFBP gene rs109669792G>A site

[0051] 1. Experimental Methods

[0052] According to the method of Example 1, the DNA samples of the 92 Chinese Holstein cows in Example 1 were PCR amplified and sequenced, and SeqMan was used to observe overlapping peaks, identify individual genotypes, and count SNP sites. Popgene software was used to calculate a series of genetic parameters for the alleles, and further calculate χ 2 value.

[0053] 2. Experimental Results

[0054] The results are shown in Table 1. The dominant allele in the rs109669792G>A site is G, and the gene frequency is 0.8913. The homozygous wild type GG is the dominant genotype, and the genotype frequency is 0.7935. The gene heterozygosity (H) of the rs109669792G>A site falls between 0.25 and 0.50, and the polymorphism information content (PIC) is less than 0.25, which indicates that this site is low polymorphic in the KIFBP gene. 2 The test results showed that the locus was in Hardy-Weinberg equilibrium in the 92 Chinese Holstein cows used (P>0.05).

[0055] Table 1 Population genetic characteristics of the rs109669792G>A locus of the KIFBP gene in Chinese Holstein dairy cows

[0056]

[0057] Note: 2 The value did not reach the significant level (P>0.05), χ 2 0.05 (df=2)>5.99(P<0.05), χ 2 0.01 (df=2)>9.21(P<0.01).

[0058] Example 3 Correlation between the rs109669792G>A site of the KIFBP gene and milk quality traits of Chinese Holstein cows

[0059] 1. Experimental Methods

[0060] The milk quality traits of the 92 Chinese Holstein cows in Example 1 were detected, including individual milk production (kg), milk fat content (%), milk protein content (%), lactose content (%), dry matter content (%), and corrected milk yield (kg), a total of 6 indicators, and the relationship between the milk quality traits and genotypes of Chinese Holstein cows was tested for significant differences, and the results were presented in the form of mean ± standard deviation.

[0061] The specific detection method is: using a milk component analyzer to detect the milk quality traits of 92 dairy cows that grew in the same environment and were in the lactation period, including: milk fat content (%), milk protein content (%), lactose content (%), and dry matter content (%).

[0062] At the same time, individual milk production, that is, the milk production of each cow in each lactation period, is the basis for statistical calculation of milk production. Individual milk production and corrected milk production are often expressed as 305-day milk production and 305-day corrected milk production.

[0063] Milk production on day 305: Collect and compile the milk production from the first day after calving to day 305. The average value is the average daily milk production of the individual.

[0064] Corrected milk production for 305 days: For lactation periods less than 305 days, or exceeding 305 days but without daily milk production records, the actual milk production recorded is multiplied by the relative coefficient and corrected to the approximate production for 305 days. The average value is the individual daily corrected milk production.

[0065] 2. Experimental Results

[0066] The results are shown in Table 2. The rs109669792G>A locus was significantly correlated with the corrected milk yield trait of Chinese Holstein cows (P<0.05). The corrected milk yield of individuals with the GG genotype was significantly higher than that of individuals with the GA genotype. There was no significant correlation with other milk quality indicators (P>0.05). There was only one AA genotype individual, which did not meet the statistical requirements and had no statistical significance.

[0067] Table 2 Correlation between rs109669792G>A site of KIFBP gene and milk quality traits of Chinese Holstein cows

[0068]

[0069] Note: The same index in the same industry with completely different letters indicates significant difference (P<0.05); the index without letters indicates no significant difference (P>0.05).

[0070] Example 4 A method for evaluating the corrected milk yield of Chinese Holstein cows

[0071] 1. Methods

[0072] The sample DNA was amplified by PCR using the primers shown in SEQ ID NOs: 1-2.

[0073] The reaction system for PCR amplification: (25 μL) template DNA 1 μL, Taq PCR Mix 12.5 μL, primers 0.7 μL each, deionized water 10.1 μL.

[0074] The reaction procedure of PCR amplification was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 5 min, and storage at 4°C for future use.

[0075] The PCR products were detected by 1.5% agarose gel electrophoresis and sequenced.

[0076] 2. Interpretation of results

[0077] The sequencing results showed that for the rs109669792 site, located at 164bp of the amplified product (SEQ ID NO: 3), located at NC_037355.1: 25247823, that is, the 25247823th nucleotide of chromosome 28 of the ARS-UCD 1.3 version of the bovine genome, there are three genotypes: AA, AG and GG. Among them, the corrected milk yield of individuals with the GG genotype was significantly higher than that of individuals with the GA genotype.

[0078] Example 5 A kit for evaluating the corrected milk yield of Chinese Holstein cows

[0079] 1. Composition

[0080] Primers shown in SEQ ID NO: 1-2, Taq PCR Mix and deionized water.

[0081] 2. Usage

[0082] Same as Example 4.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for evaluating the corrected milk yield of Chinese Holstein cows, characterized in that: The genotype of the rs109669792 locus was detected; the rs109669792 locus was located at NC_037355.1:25247823, i.e., position 25247823 of chromosome 28 of the ARS-UCD 1.3 version of the bovine genome, and there were three genotypes: AA, AG and GG, among which the corrected milk yield of individuals with the GG genotype was significantly higher than that of individuals with the GA genotype.

2. The method according to claim 1, characterized in that The sample DNA was amplified by PCR using the primers shown in the nucleotide sequences SEQ ID NOs: 1-2 to detect the genotype of rs109669792.

3. The method according to claim 2, characterized in that The reaction system of the PCR amplification is: 1 μL of template DNA, 12.5 μL of Taq PCR Mix, 0.7 μL of each primer, and 10.1 μL of deionized water.

4. The method according to claim 2, characterized in that: The reaction procedure of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, 40 cycles; 72°C for 5 min.

5. A primer for evaluating the corrected milk yield of Chinese Holstein cows, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NOs: 1-2.

6. Application of reagents for detecting the genotype of rs109669792 in evaluating the corrected milk yield of Chinese Holstein cows.

7. Use of a reagent for detecting the genotype of rs109669792 in the preparation of a kit for evaluating the corrected milk yield of Chinese Holstein cows.

8. A kit for evaluating the corrected milk yield of Chinese Holstein cows, characterized in that: Contains reagents for detecting the genotype of rs109669792.

9. A method for breeding Chinese Holstein cows with high corrected milk yield, characterized in that: The method described in claim 1 is used to select Chinese Holstein cows with high corrected milk yield as parents for breeding.

10. Use of the method according to claim 1, the primer according to claim 5 or the method according to claim 9 in molecular breeding of Chinese Holstein cows with high corrected milk yield.

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