A method for evaluating daily average milk yield and / or corrected milk yield in milk quality traits of Chinese Holstein cows

Abstract

The application discloses a method for evaluating daily average milk yield and / or corrected milk yield of Chinese Holstein cow milk quality traits. The application finds that a rs382915208 site of a TFAP4 gene fragment, namely, an I6-653G>C site, is located at NC_037352.1:3444953, namely, 3444953 of chromosome 25 of a bovine genome ARS-UCD version 2.0, and there are three genotypes of GG, CG and CC. The milk yield and the corrected milk yield of the GG genotype are significantly higher than those of the CC type. The rs382915208 site is used to establish a method and a kit for evaluating Chinese Holstein cow milk quality traits, and the method and the kit can be applied to molecular breeding of Chinese Holstein cows.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, in particular, to a method for evaluating Chinese Holstein cow milk quality traits of daily average milk yield and / or corrected milk yield. BACKGROUND

[0002] Chinese Holstein cow, also known as Chinese black and white spotted cow, is a breed developed by crossbreeding Holstein cow introduced into China at the end of the 19th century with Chinese yellow cow, and gradually formed through continuous improvement. It is also a widely used dairy breed in China.

[0003] Currently, it has been found that multiple major genes such as ACADSB, PRL, ANGPTL5, MASP2, C3, LBP, PDE10A, SERPINE2, and CXCR2 are related to milk quality traits.

[0004] Transcription factor activating enhancer-binding protein 4 (TFAP4) is a transcriptional regulator and a member of the basic helix-loop-helix transcription factor family. It is not only related to normal development, but also seems to be related to cell tumor formation. Silencing endogenous TFAP4 can inhibit the malignant proliferation of endometrial cancer cells in an in vitro model. The mechanism may be that it disrupts the transcriptional repression of CDKN1A by the TFAP4 / Pokemon transcription complex, thereby suppressing the cycle of tumor cells. TFAP4 protein is highly expressed in non-small cell lung cancer tissues. Down-regulation of TFAP4 gene expression in A549 cells can inhibit cell proliferation, migration, and invasion. c-MYC limits the self-renewal of proliferating B lymphocytes by inducing TFAP4, which unexpectedly inhibits the occurrence of lymphocyte cancer. TFAP4 transcriptionally activates BBOX1-AS1, which is highly expressed in gastric cancer tissues and cell lines, thereby promoting the proliferation and migration of gastric cancer cells MKN45. TFAP2B is specifically highly expressed in human epidermal melanocytes, and its expression is significantly positively correlated with the expression of DCT, a marker molecule of melanocytes, and is significantly lower in the epidermal melanocytes of patients with vitiligo. TFAP2C plays a pro-cancer role in bladder cancer, and its up-regulated expression level is associated with poor prognosis.

[0005] It can be seen that TFAP4 is involved in controlling the proliferation and differentiation of various cells. Currently, the research objects of TFAP4 in the industry mainly focus on the human body, and there are few or even no related investigations and researches in the agricultural field. SUMMARY

[0006] The present application aims to overcome the above-mentioned deficiencies in the prior art, and provides a method for evaluating the average daily milk yield and / or corrected milk yield of Chinese Holstein dairy cows.

[0007] The first object of the present application is to provide a method for evaluating the milk quality traits of Chinese Holstein dairy cows.

[0008] The second object of the present application is to provide a kit for evaluating the milk quality traits of Chinese Holstein dairy cows.

[0009] The third object of the present application is to provide the use of a reagent for detecting the genotype of the SNPs site I6-653 G>C in the evaluation of the milk quality traits of Chinese Holstein dairy cows.

[0010] The fourth object of the present application is to provide the use of a reagent for detecting the genotype of the SNPs site I6-653 G>C in the preparation of a kit for evaluating the milk quality traits of Chinese Holstein dairy cows.

[0011] The fifth object of the present application is to provide the use of the method or the kit in the kit for evaluating the milk quality traits of Chinese Holstein dairy cows.

[0012] In order to achieve the above-mentioned objects, the present application is realized by the following scheme:

[0013] The present application claims a method for evaluating the milk quality traits of Chinese Holstein dairy cows, which detects the genotype of the SNPs site I6-653 G>C, i.e. rs382915208 site, located at NC_037352.1:3444953, i.e. the 3444953th position of chromosome 25 of the ARS-UCD 2.0 version of bovine genome, which has three genotypes of GG, CG and CC.

[0014] Preferably, the average daily milk yield of the GG genotype individual is significantly higher than that of the CC genotype individual; and the corrected milk yield of the GG genotype individual is significantly higher than that of the CC genotype individual.

[0015] Preferably, the genotype of the SNPs site I6-653 G>C is detected by PCR amplification using nucleotide sequences such as primers of SEQ ID NO:1-2.

[0016] The application also claims a kit for evaluating milk quality traits of Chinese Holstein cows, containing reagents for detecting the genotype of the SNPs site I6-653 G>C, i.e. rs382915208, located at NC_037352.1:3444953, i.e. the 25th chromosome of the ARS-UCD 2.0 version of the bovine genome, at position 3444953, with three genotypes of GG, CG and CC.

[0017] Preferably, the daily average milk yield of the GG genotype individual is significantly higher than that of the CC genotype individual; the corrected milk yield of the GG genotype individual is significantly higher than that of the CC genotype individual.

[0018] Preferably, the reagent is a nucleotide sequence such as the primers of SEQ ID NO:1-2.

[0019] The upstream primer sequence is 5'-GGCTGTCCTTTGTTCCTGG-3'(SEQ ID NO:1),

[0020] The downstream primer sequence is 5'-CACTCATTCACCCTCGTCTC-3'(SEQ ID NO:2).

[0021] The application also claims the following applications:

[0022] The reagent for detecting the genotype of the SNPs site I6-653 G>C in the application of evaluating milk quality traits of Chinese Holstein cows.

[0023] The reagent for detecting the genotype of the SNPs site I6-653 G>C in the application of preparing a kit for evaluating milk quality traits of Chinese Holstein cows.

[0024] The method or the kit in the application of the kit for evaluating milk quality traits of Chinese Holstein cows.

[0025] Preferably, the milk quality trait is daily average milk yield or corrected milk yield.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] The application discovers that the SNP site I6-653 G>C in the intron of the TFAP4 gene fragment, that is, the rs382915208 site, is located at NC_037352.1:3444953, that is, the 3444953th position of chromosome 25 of the ARS-UCD version 2.0 bovine genome, and there are three genotypes of GG, CG and CC. The milk yield and corrected milk yield of the GG genotype are significantly higher than those of the CC type. A method and kit for evaluating the milk quality traits of Chinese Holstein cows are established by using the SNP site I6-653 G>C, which can be applied to the molecular breeding of Chinese Holstein cows. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the electrophoresis result of the mixed pool PCR amplification of the TFAP4 gene of Chinese Holstein cows; M. DL-2 000Marker; the lane 1 is the PCR amplification product of the mixed pool.

[0029] Figure 2 It is the sequencing result of the target product of the mixed pool PCR amplification of the TFAP4 gene of Chinese Holstein cows.

[0030] Figure 3 It is the electrophoresis result of the individual PCR amplification of the TFAP4 gene of Chinese Holstein cows; M. DL-2 000Marker; the lanes 1-3 are respectively the PCR amplification products of different individuals.

[0031] Figure 4 It is the sequencing peak chart result of the three genotypes of the I6-653 G>C site of the TFAP4 gene; a is the individual of the CC genotype; b is the individual of the GC genotype; c is the individual of the GG genotype. DETAILED DESCRIPTION

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

[0033] Example 1 Amplification of the TFAP4 gene

[0034] I. Experimental samples

[0035] 110 adult healthy Chinese Holstein cows in the same growth period and under the same feeding conditions were selected, 10 mL of blood was taken from each through the jugular vein, and after anticoagulation treatment, it was properly stored at minus 80°C.

[0036] II. Experimental method

[0037] 1. Primer design

[0038] Primers were designed based on the TFAP4 gene DNA sequence (ID: ENSBTAT00000047117.5) provided in the Ensembl database, and the primer sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Guangzhou). The primer sequences are as follows:

[0039] The upstream primer sequence is: 5'-GGCTGTCCTTTGTTCCTGG-3' (SEQ ID NO:1),

[0040] The downstream primer sequence is: 5'-CACTCATTCACCCTCGTCTC-3' (SEQ ID NO:2).

[0041] 2. Mixed-pool PCR amplification and sequencing

[0042] Blood genomic DNA samples from 32 randomly selected Chinese Holstein dairy cows were mixed in equal volumes to prepare a DNA pool sample, which was then amplified by PCR using primers shown in SEQ ID NO:1-2.

[0043] PCR amplification system (total volume 25 μL): 12.5 μL of 2×Green Taq Mix, 10.1 μL of ddH2O, 0.7 μL each of the primers shown in SEQ ID NO: 1-2, and 1 μL of template.

[0044] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 48 s, 35 cycles; 72℃ final extension for 5 min, store at 4℃.

[0045] PCR products were verified by electrophoresis on a 1.0% agarose gel. If the target band on the electrophoresis gel matched the target fragment of the primers, and the band was single and bright, the product could be sent to Beijing Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing.

[0046] 3. Analysis of mixed-pool sequencing results

[0047] SeqMan software was used to analyze the mixed-pool sequencing results and sequencing peak diagrams to identify double peaks and determine SNP sites.

[0048] II. Experimental Results

[0049] Electrophoresis results as follows Figure 1 As shown, the PCR amplification product, when detected by 1.0% agarose gel electrophoresis, showed a target band length of 841 bp, consistent with the expected target fragment size. The band was single, well-imaged, and showed no non-specific bands, making it suitable for sequencing. The product sequence is SEQ ID NO: 3, i.e.:

[0050]

[0051] (underlined are primers shown in SEQ ID NO: 1-2, boxed The locus is labeled rs382915208. )。

[0052] Sequencing by Figure 2 It can be seen that after the mixed-pool PCR amplification products were sent to a third-party company for sequencing, the sequencing results and peak diagrams were observed using SeqMan software to look for double peaks. The results showed that at the 653rd base of the sixth intron, i.e., at 414bp of the PCR product, a mutation from C to G was found. This SNP site is I6-653 G>C, i.e., rs382915208, located at NC_037352.1:3444953, which is the 3444953rd position on chromosome 25 of the bovine genome in ARS-UCD 2.0. There are three genotypes: GG, CG, and CC.

[0053] Example 2: Population genetic characteristics of TFAP4 gene SNPs in Chinese Holstein dairy cattle

[0054] I. Experimental Methods

[0055] 1. Individual PCR amplification and sequencing

[0056] Individual PCR amplification was performed using the primers shown in SEQ ID NO:1-2 as templates from the blood DNA of all 110 Chinese Holstein dairy cows in Example 1.

[0057] The PCR amplification system and procedure were the same as in Example 1. Subsequently, the individual PCR products were placed in a 1.0% agarose gel for electrophoresis verification. Then, the individual PCR stock solution with a single, bright band that matched the target fragment of the primers was sent to Beijing Qingke Biotechnology Co., Ltd. (Guangzhou) for sequencing.

[0058] 2. Data Statistics and Analysis

[0059] Seqman software was used to analyze the sequencing results and peak plots of each individual to identify mutation sites. SNP loci, genotypes, gene frequencies, and genotype frequencies for each individual were statistically analyzed. Heterozygosity (H) was calculated using gene and genotype frequencies. The LSD method in SPSS 23.0 statistical software was used to analyze the correlation between different genotypes and haplotypes and milk quality traits in Chinese Holstein dairy cows. Results are expressed as mean ± standard error; P < 0.05 indicates significant difference, and P < 0.01 indicates highly significant difference.

[0060] II. Experimental Results

[0061] Electrophoresis results as follows Figure 3 As shown, the individual PCR amplification products were detected by 1.0% agarose gel electrophoresis. The target band length was 841 bp, which was consistent with the expected fragment and the band size of the mixed pool PCR amplification products. The band was single, the imaging was good, and there were no non-specific bands, so it could be used for sequencing.

[0062] Using SeqMan software, the sequencing results and peak diagrams of PCR from 110 individual Holstein dairy cows in China were compared and analyzed sequentially. The sequencing results are as follows: Figure 4 As shown, there is one SNP site in the target band of the TFAP4 gene, namely I6-653G>C. Among them, I6-653G>C has three genotypes: CC, GC, and GG.

[0063] The analysis results of the I6-653 G>C locus are shown in Table 1. The dominant genotype at the I6-653 G>C locus is CC, accounting for approximately 60%, with C being the dominant allele and a gene frequency of 0.786. The heterozygosity (H) of the I6-653 G>C locus is 0.37, and the polymorphism information content (PIC) is 0.28, both falling within the range of 0.25 to 0.5, indicating that this locus exhibits moderate polymorphism in the population. The chi-square test results show that the χ² value of this SNP... 2 The value is 1.32, which is less than χ. 2 0.05 (χ 2 0.05 =5.99), P>0.05, indicating that this gene locus is in Hardy-Weinberg equilibrium in the Chinese Holstein dairy cow population.

[0064] Table 1. Population genetic characteristics analysis results of TFAP4 gene SNPs in Chinese Holstein dairy cattle.

[0065]

[0066] Note: χ 2 The value indicates that the significance level was not reached (P>0.05), χ² 2 0.05 (df=2)=5.99, χ 2 0.01 (df=2)=9.21

[0067] Example 3: Association Analysis of Different SNP Genotypes with Milk Quality Traits in Chinese Holstein Cows

[0068] I. Experimental Methods

[0069] The milk quality traits of 110 adult healthy Chinese Holstein dairy cows in Example 1 were detected and recorded. The specific indicators included: milk yield, corrected milk yield, milk fat content (milk fat percentage), milk protein content (milk protein percentage), lactose content, dry matter content, and urea nitrogen content.

[0070] The specific testing method was as follows: The milk quality traits of 110 dairy cows that were growing in the same environment and were in the lactation period were tested using a milk composition analyzer, including five indicators: milk fat content (%), milk protein content (%), lactose content (%), urea nitrogen content (mg / L), and dry matter content (%).

[0071] Meanwhile, individual milk production, i.e., the milk production of each dairy cow in each lactation period, is the basis for milk production statistics. Individual milk production and corrected milk production are often expressed as milk production over 305 days and corrected milk production over 305 days.

[0072] Milk production over 305 days: Milk production was collected and statistically analyzed from the first day after calving to the 305th day. The average daily milk production per calf was calculated.

[0073] Corrected milk production over 305 days: For lactation periods less than 305 days, or exceeding 305 days without daily milk production records, the actual milk production from these records is multiplied by a relative coefficient to correct for an approximate daily production over 305 days. The average value is then calculated as the individual's corrected daily milk production.

[0074] II. Experimental Results

[0075] The results are shown in Table 2. The genotype at the I6-653 G>C locus mainly affects milk production. For example, the average daily milk production of individuals with the GG genotype (32.44 kg) was significantly higher than that of the dominant CC genotype (25.83 kg) (P<0.05), about 25% higher. The corrected milk production of the GG genotype (52.31 kg) was also significantly higher than that of the CC genotype (39.03 kg) (P<0.05), up to 34% higher.

[0076] Therefore, even though the average daily milk yield of the GC genotype (26.11 kg) is slightly higher than that of the CC genotype, although the difference is not significant, it is still evident that allele G plays a positive role in milk yield in Chinese Holstein cattle.

[0077] Regarding other milk quality indicators, there were no significant differences among genotypes at the I6-653 G>C locus, indicating no correlation (P>0.05).

[0078] Table 2. Association analysis of TFAP4 gene SNP loci with milk quality traits in Chinese Holstein dairy cows.

[0079] Genotype GC GG CC Milk yield (kg) 26.11±6.11 32.44 ± 4.93 b ]] 25.83 ± 5.30 a ]] Fat content of milk (%) 4.60±0.39 4.39±0.25 4.90±2.50 Protein content of milk (%) 3.47±0.21 3.31±0.11 3.47±0.23 Lactose content of milk (%) 4.75±0.18 4.84±0.27 4.77±0.15 Dry matter content of milk (%) 14.00±1.56 13.10±0.55 14.08±2.11 Urea nitrogen content (mg / L) 19.58±1.59 18.77±1.95 19.51±1.58 Corrected milk yield (kg) 40.78±10.27 52.31 ± 5.87 b ]] 39.03 ± 10.65 a ]]

[0080] Example 4: A method for evaluating milk quality traits of Chinese Holstein dairy cows

[0081] I. Methods

[0082] DNA from Chinese Holstein dairy cows was amplified by PCR using primers with nucleotide sequences such as SEQ ID NO:1-2.

[0083] PCR amplification system (total volume 25 μL): 12.5 μL of 2×Green Taq Mix, 10.1 μL of ddH2O, 0.7 μL each of the primers shown in SEQ ID NO: 1-2, and 1 μL of template DNA.

[0084] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 45 s, 72℃ extension for 48 s, 35 cycles; 72℃ final extension for 5 min, store at 4℃.

[0085] The PCR products were verified by electrophoresis on a 1.0% agarose gel. If the target band was single and bright, sequencing could proceed.

[0086] II. Result Interpretation

[0087] The I6-653 G>C locus, also known as rs382915208, is located at NC_037352.1:3444953, which corresponds to position 3444953 on chromosome 25 of the bovine genome in ARS-UCD2.0. It contains three genotypes: GG, CG, and CC. Individuals with the GG genotype have significantly higher average daily milk production than those with the CC genotype; the corrected milk production of individuals with the GG genotype is also significantly higher than that of individuals with the CC genotype.

[0088] Example 5: A kit for evaluating milk quality traits of Chinese Holstein cows

[0089] I. Composition

[0090] Using nucleotide sequences such as SEQ ID NO:1-2 primers, 2×Green Taq Mix, and 10.1 μL of ddH2O.

[0091] II. Instructions for Use

[0092] Same as Example 4.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for evaluating milk quality traits in Chinese Holstein cows, characterized by, Detecting the genotype of SNP site rs382915208; the daily average milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype; the corrected milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype.

2. The method of claim 1, wherein, The genotype of rs382915208 is detected by PCR amplification using primers with nucleotide sequences as shown in SEQ ID NO: 1-2.

3. Use of a reagent containing the genotype of SNP site rs382915208 in the preparation of a kit for evaluating milk quality traits of Chinese Holstein cows, characterized in that, The daily average milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype; the corrected milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype.

4. Use 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 use of a reagent for detecting the genotype of SNP site rs382915208 in the evaluation of milk quality traits of Chinese Holstein cows, characterized in that, The daily average milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype; the corrected milk yield of the individual with GG genotype of the SNP site is significantly higher than that of the individual with CC genotype.

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