Method for evaluating average daily milk yield and / or correcting milk yield in milk quality traits of Chinese Holstein cows

Abstract

The invention discloses a method for evaluating daily average milk yield and / or corrected milk yield in Chinese Holstein cow milk quality traits, which discovers that an rs382915208 site, namely an I6-653G > C site, located on a TFAP4 gene segment is located on the 3444953 site of the 25th chromosome of NC037352.1: 3444953, namely an AR-UCD 2.0 edition cow genome, has three genotypes, namely GG, CG and CC, and is used for evaluating the daily average milk yield and / or corrected milk yield of the Chinese Holstein cow milk quality traits. The milk yield and corrected milk yield of the GG genotype are obviously higher than those of the CC genotype. The rs382915208 site is utilized to establish a method and a kit for evaluating the milk quality character of the Chinese Holstein dairy cow, and the method and the kit can be applied to molecular breeding of the Chinese Holstein dairy cow.

Description

Technical Field

[0001] The present invention relates to the field of molecular breeding technology, and in particular to a method for evaluating the average daily milk yield and / or corrected milk yield in milk quality traits of Chinese Holstein cows. Background Art

[0002] Chinese Holstein cows, also known as Chinese black and white dairy cows, are a breed bred in the late 19th century by crossbreeding Holstein cows introduced to China at that time with Chinese yellow cattle. It has gradually formed through continuous improvement and is now a widely used dairy cow breed in my country.

[0003] It has been found that several major effect genes such as ACADSB, PRL, ANGPTL5, MASP2, C3, LBP, PDE10A, SERPINE2, and CXCR2 are correlated with milk quality traits.

[0004] Transcription factor activating enhancer-binding protein 4 (TFAP4), as a transcriptional regulatory factor, is 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, and its mechanism may be related to the destruction of the transcriptional inhibition of CDKN1A by the TFAP4 / Pokemon transcription complex, thereby inhibiting the cycle of tumor cells; TFAP4 protein is highly expressed in non-small cell lung cancer tissues, and downregulating TFAP4 gene expression in A549 cells can inhibit cell proliferation , migration and invasion ability; c-MYC inducing TFAP4, limiting the self-renewal of proliferating B lymphocytes, unexpectedly inhibiting the occurrence of lymphocytic carcinoma; 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 cell MKN45; TFAP2B is specifically highly expressed in human epidermal melanocytes, and its expression is significantly positively correlated with the expression of melanocyte marker molecule DCT, and is significantly lowly expressed in epidermal melanocytes of vitiligo patients; TFAP2C plays a carcinogenic role in bladder cancer, and its upregulated 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. At present, the research object of TFAP4 in the industry is mainly focused on the human body, and there are relatively few or even no related investigations and studies in the agricultural field. Summary of the invention

[0006] 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 average daily milk yield and / or corrected milk yield in the milk quality traits of Chinese Holstein cows.

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

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

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

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

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

[0012] In order to achieve the above object, the present invention is implemented by the following scheme:

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

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

[0015] Preferably, the nucleotide sequence such as SEQ ID NO: 1-2 primers are used for PCR amplification to detect the genotype of the SNPs site I6-653 G>C.

[0016] The present invention also claims a kit for evaluating the milk quality traits of Chinese Holstein cows, comprising a reagent for detecting the genotype of the SNPs site I6-653 G>C, wherein the SNPs site is I6-653 G>C, i.e., the rs382915208 site, located at NC_037352.1:3444953, i.e., the 3444953rd position of chromosome 25 of the ARS-UCD 2.0 version of the bovine genome, and there are three genotypes: GG, CG and CC.

[0017] Preferably, the average daily milk production of individuals with the GG genotype is significantly higher than that of individuals with the CC genotype; and the corrected milk production of individuals with the GG genotype is significantly higher than that of individuals with the CC genotype.

[0018] Preferably, the reagent is a nucleotide sequence such as a primer 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 present invention also claims the following applications:

[0022] Application of reagents for detecting the genotype of SNPs locus I6-653 G>C in evaluating milk quality traits of Chinese Holstein cows.

[0023] Application of a reagent for detecting the genotype of SNPs locus I6-653 G>C in the preparation of a kit for evaluating milk quality traits of Chinese Holstein cows.

[0024] Application of the method or the kit in a kit for testing milk quality traits of Chinese Holstein cows.

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

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

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

[0028] Figure 1 The electrophoresis results of PCR amplification of the TFAP4 gene in the mixed pool of Chinese Holstein cows; M.DL-2 000 Marker; Lane 1 is the PCR amplification product of the mixed pool.

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

[0030] Figure 3 The electrophoresis results of PCR amplification of TFAP4 gene in Chinese Holstein cows; M.DL-2 000 Marker; Lanes 1 to 3 are the PCR amplification products of different individuals.

[0031] Figure 4 The results of sequencing peaks of three genotypes at the I6-653 G>C site of the TFAP4 gene; a is an individual with CC genotype; b is an individual with GC genotype; c is an individual with GG genotype. 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 Amplification of TFAP4 gene

[0034] 1. Experimental Sample

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

[0036] 2. Experimental Methods

[0037] 1. Primer design

[0038] According to the TFAP4 gene DNA sequence provided by the Ensembl database (number: ENSBTAT00000047117.5), primers were designed and 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. PCR amplification and sequencing of mixed pools

[0042] Blood genomic DNA samples from 32 Chinese Holstein cows were randomly selected and mixed in equal amounts to prepare a DNA pool sample, and then PCR amplification was performed using the primers shown in SEQ ID NOs: 1-2.

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

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

[0045] The PCR product was verified by electrophoresis on 1.0% agarose gel. If the target band of electrophoresis is consistent with the target fragment of the primer, and the band is single and bright, it can 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 graphs to find double peaks and determine SNP sites.

[0048] 2. Experimental Results

[0049] The electrophoresis results are as follows Figure 1As shown, the PCR amplification product was detected by 1.0% agarose gel electrophoresis, and the target band length was observed to be 841 bp, which was consistent with the expected target fragment size, and the band was single, the imaging was good, and there was no non-specific band, which could be used for sequencing. The product sequence is SEQ ID NO: 3, that is:

[0050]

[0051] (The underlined primers are shown in SEQ ID NO: 1-2, the boxed Annotated as rs382915208 site )。

[0052] Sequencing by Figure 2 It can be seen that after the mixed 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. It was found that at the 653rd base of the sixth intron, that is, 414bp of the PCR product, a mutation occurred from C to G. This SNPs site is I6-653 G>C, that is, the rs382915208 site, located at NC_037352.1:3444953, that is, the 3444953rd position of chromosome 25 of the ARS-UCD 2.0 version of the bovine genome, and there are three genotypes: GG, CG and CC.

[0053] Example 2 Population genetic characteristics of TFAP4 gene SNPs in Chinese Holstein cows

[0054] 1. Experimental Methods

[0055] 1. Individual PCR amplification and sequencing

[0056] Individual PCR amplification was performed using the primers shown in SEQ ID NOs: 1-2 with the blood samples DNA of all 110 Chinese Holstein cows in Example 1 as templates.

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

[0058] 2. Statistics and analysis of data

[0059] Seqman software was used to analyze the results and peak graphs of individual sequencing in turn to find the mutation sites, and the SNP sites and genotypes, gene frequencies, and genotype frequencies of each individual were statistically analyzed. The genetic heterozygosity H (heterozygosity) was calculated by gene and genotype frequencies; the LSD method of SPSS23.0 statistical software was used to analyze the correlation between different genotypes and different haplotypes and the milk quality traits of Chinese Holstein cows. The results were expressed as "mean ± standard error", P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0060] 2. Experimental Results

[0061] The electrophoresis results are as follows Figure 3 As shown, the individual PCR amplification products were detected by 1.0% agarose gel electrophoresis, and the target band length was 841 bp, which was consistent with the expected fragment and the band size of the mixed pool PCR amplification product. The band was single and the imaging was good, without non-specific bands, and could be used for sequencing.

[0062] Using SeqMan software, the sequencing results and peak graphs of PCR of 110 Chinese Holstein cows were compared and analyzed in turn. Figure 4 As shown, there is a total of 1 SNPs site in the target band of TFAP4 gene, namely I6-653G>C. Among them, I6-653 G>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 in the I6-653 G>C locus is CC, accounting for about 60%, and C is its dominant allele with a gene frequency of 0.786. The gene heterozygosity (H) of I6-653 G>C is 0.37, and the polymorphic information content (PIC) is 0.28, both between 0.25 and 0.5, indicating that the locus is moderately polymorphic in the population. According to the chi-square test results, the χ2 of this SNPs is 2 The value is 1.32, which is less than χ 2 0.05 (x 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 Results of population genetic characteristics analysis of TFAP4 gene SNPs in Chinese Holstein dairy cows

[0065]

[0066] Note: 2 The value did not reach the significant level (P>0.05), χ 20.05 (df ​​= 2) = 5.99, χ 2 0.01 (df=2)=9.21

[0067] Example 3 Analysis of association between different genotypes of SNPs and milk quality traits of Chinese Holstein cows

[0068] 1. Experimental Methods

[0069] The milk quality trait-related indicators of the 110 adult healthy Chinese Holstein cows of Example 1 were detected and recorded, and the specific indicators included: milk yield, corrected milk yield, milk fat content (milk fat rate), milk protein content (milk protein rate), lactose content, dry matter content, urea nitrogen content, etc.

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

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 2. Experimental Results

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

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

[0077] As for other milk quality indicators, there was no significant difference among the genotypes at the I6-653 G>C site, and there was no correlation (P>0.05).

[0078] Table 2 Association analysis between genotypes of TFAP4 gene SNP loci and milk quality traits of Chinese Holstein cows

[0079] genotype GC GG CC Milk production (kg) 26.11±6.11 <![CDATA[32.44±4.93 b ]]> <![CDATA[25.83±5.30 a ]]> Milk fat content (%) 4.60±0.39 4.39±0.25 4.90±2.50 Milk protein content (%) 3.47±0.21 3.31±0.11 3.47±0.23 Lactose content (%) 4.75±0.18 4.84±0.27 4.77±0.15 Dry matter content (%) 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 <![CDATA[52.31±5.87 b ]]> <![CDATA[39.03±10.65 a ]]>

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

[0081] 1. Methods

[0082] PCR amplification of Chinese Holstein cow DNA was performed using primers such as nucleotide sequences such as SEQ ID NO: 1-2.

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

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

[0085] The PCR product was verified by electrophoresis on 1.0% agarose gel. If the target band of electrophoresis is single and bright, it can be sequenced.

[0086] 2. Interpretation of results

[0087] The I6-653 G>C site, i.e., rs382915208 site, is located at NC_037352.1:3444953, i.e., position 3444953 of chromosome 25 of the ARS-UCD2.0 version of the bovine genome, and there are three genotypes: GG, CG, and CC. The average daily milk production of individuals with the GG genotype is significantly higher than that of individuals with the CC genotype; the corrected milk production of individuals with the GG genotype is 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] 1. Composition

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

[0091] 2. Usage

[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 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 milk quality traits of Chinese Holstein cows, characterized in that: The genotype of the detected SNPs site is I6-653G>C. The SNPs site is I6-653 G>C, that is, the rs382915208 site, located at NC_037352.1:3444953, that is, the 3444953rd position of chromosome 25 of the ARS-UCD 2.0 version of the bovine genome, and there are three genotypes: GG, CG and CC.

2. The method according to claim 1, characterized in that The average daily milk production of individuals with GG genotype was significantly higher than that of individuals with CC genotype; the corrected milk yield of individuals with GG genotype was significantly higher than that of individuals with CC genotype.

3. The method according to claim 1 or 2, characterized in that: The nucleotide sequence such as SEQ ID NO: 1-2 primers are used for PCR amplification to detect the genotype of the SNPs site I6-653 G>C.

4. A kit for evaluating the milk quality traits of Chinese Holstein cows, characterized in that: The invention contains a reagent for detecting the genotype of the SNPs site I6-653 G>C, wherein the SNPs site is I6-653 G>C, i.e., the rs382915208 site, located at NC_037352.1:3444953, i.e., the 3444953rd position of the 25th chromosome of the ARS-UCD 2.0 version of the bovine genome, and there are three genotypes: GG, CG and CC.

5. The kit according to claim 4, characterized in that The average daily milk production of individuals with GG genotype was significantly higher than that of individuals with CC genotype; the corrected milk yield of individuals with GG genotype was significantly higher than that of individuals with CC genotype.

6. The kit according to claim 4 or 5, characterized in that The reagent is a nucleotide sequence such as SEQ ID NO: 1-2 primer.

7. Application of reagents for detecting the genotype of SNPs locus I6-653 G>C in evaluating milk quality traits of Chinese Holstein cows.

8. Application of a reagent for detecting the genotype of the SNPs locus I6-653 G>C in the preparation of a kit for evaluating milk quality traits of Chinese Holstein cows.

9. Use of the method according to claim 1 or the kit according to claim 4 in a kit for testing milk quality traits of Chinese Holstein cows.

10. The use according to claim 9, characterized in that: The milk quality trait is average daily milk yield or corrected milk yield.

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