SNP (Single Nucleotide Polymorphism) molecular marker for improving milk yield of Holstein cattle and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker for increasing the milk yield of Holstein cattle and application of the SNP molecular marker, the SNP molecular marker is located at a mutation site of a Holstein cattle genome Leptin gene, the gene number of the Leptin gene in an NCBI database is NC037333.1, the SNP molecular marker is located at the 1761th basic group of a cDNA sequence of the Leptin gene, and the mutation basic group is A or G. When the basic group of the SNP molecular marker is A, the genotype is AA or AG, and the milk fat rate and the milk protein rate of Holstein cow milk production are high; when the basic group of the SNP molecular marker is G, the genotype is GG, the milk yield of Holstein cattle in 305 days is high, and the milk quality is not obviously reduced. The marker is applied to genetic improvement of dairy cows, and the frequency of dominant alleles can be improved generation by generation by selecting individuals carrying the dominant alleles, so that the lactation performance of the dairy cows is effectively improved, the breeding efficiency is optimized, and the competitive advantage in the field of dairy cow breeding is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to a SNP molecular marker for improving the milk yield of Holstein cows and its application, belonging to the technical field of genes. BACKGROUND

[0002] With the rapid advancement of the intensive and large-scale dairy farming, improving the milk performance of dairy cows has become one of the key technical targets for improving the yield and quality of dairy products and accelerating the efficiency of genetic improvement. The lactation performance of dairy cows is a typical multi-factor complex trait, in which the genetic factor plays a dominant role in its formation and expression. The fat content (FC) and protein content (PC) in milk are not only the core indicators for measuring the nutritional value and processing adaptability of dairy products, but also have a direct impact on the commercial value of dairy products. These traits generally have a medium or above genetic heritability, and can be effectively improved through genetic selection.

[0003] The existing traditional breeding method mainly relies on artificial determination and intergenerational selection of phenotypic traits, but this method is greatly affected by environmental interference and progresses slowly, which is difficult to meet the actual needs of efficient selection. With the in-depth development of molecular genetics and genomics, the molecular assisted selection (MAS) technology based on genetic markers has been widely applied in the high-yield selection process of dairy cows. A large number of studies have shown that a plurality of functional genes and single nucleotide polymorphism (SNP) sites closely related to lactation traits can be used as genetic markers for molecular screening. Therefore, exploring and utilizing SNP molecular markers significantly related to key lactation traits such as fat content and protein content has become an important technical approach to realize the high-yield and high-quality selection of dairy cows.

[0004] Leptin is a peptide hormone with a wide range of biological activities, which is mainly synthesized by white adipose tissue. Its circulating concentration is positively correlated with the level of fat storage in the body, and is regulated by energy intake. Short-term fasting can reduce its expression. Dairy cow mammary glands are derived from adipose tissue and have the ability to secrete Leptin. Leptin is expressed in the mammary gland and its receptors are widely present. Studies have shown that Leptin and its receptors are expressed in mammary gland tissue and may be involved in the activation and differentiation of mammary gland cells. Genetic studies have shown that SNPs in the Leptin and its receptor genes can affect the growth and development, milk production performance and reproductive capacity of ruminants. As a gene that plays a key role in the regulation of sugar, fat and energy metabolism, SNPs mutation in the Leptin gene may regulate gene expression by affecting its transcription activity or mRNA stability, ultimately affecting the lactation capacity and milk component synthesis of dairy cows. Therefore, studying the potential functional SNP sites in the sequence of the Leptin gene has a clear value in molecular breeding. SUMMARY

[0005] The application aims to provide a SNP molecular marker for improving milk yield of Holstein cows and application thereof.

[0006] The SNP molecular marker for improving milk yield of Holstein cows is located at a mutation site of a Leptin gene in a Holstein cow genome, the Leptin gene has a gene number of NC_037333.1 in a NCBI database, and the SNP molecular marker is located at a base at position 1761 of a Leptin gene cDNA sequence, and the mutation base is A or G.

[0007] Further, when the SNP molecular marker base is A, the genotype is AA or AG, the milk fat rate and milk protein rate of the Holstein cow are high, and when the SNP molecular marker base is G, the genotype is GG, and the 305-day milk yield of the Holstein cow is high.

[0008] The primer pair for amplifying the SNP molecular marker is as shown in SEQ ID NO. 1-3.

[0009] The reagent or kit containing the primer pair is provided.

[0010] Further, the kit further comprises KASPMaster Mix.

[0011] The SNP molecular marker, the primer pair or the reagent or kit is applied to detection of lactation performance of the Holstein cow or Holstein cow breeding.

[0012] Further, the method for detecting the lactation performance of the Holstein cow or the Holstein cow breeding comprises the following steps.

[0013] (1) extracting Holstein cow blood genomic DNA as template DNA;

[0014] (2) using specific primers, performing PCR amplification on the genomic DNA of the Holstein cow to be detected obtained in step (1) to obtain a PCR amplification product;

[0015] (3) purifying the PCR amplification product obtained in step (2) to perform genotyping detection, when the SNP molecular marker base is A, the genotype is AA or AG, the milk fat rate and milk protein rate of the Holstein cow are high, and when the SNP molecular marker base is G, the genotype is GG, and the 305-day milk yield of the Holstein cow is high.

[0016] Further, the PCR amplification system comprises 1.25 μL of template DNA, 0.625 μL of each of upstream and downstream primers, and 2.5 μL of KASP Master Mix.

[0017] Further, the PCR amplification procedure is 95℃ for 15 min, 94℃ for 20 s, 61℃ for 60 s for 10 cycles, 94℃ for 20 s, 55℃ for 60 s for 26 cycles, and 37℃ for 1 min.

[0018] Beneficial effects: Compared with the prior art, the application has the following remarkable advantages: the SNP molecular marker for improving the milk yield of Holstein cows provided by the application is located on chromosome 4 of the Holstein genome, and is related to the mutation of the Leptin gene. Specifically, the SNP molecular marker is located at the 1761th site of the Leptin gene, and has G / A polymorphism. When the SNP molecular marker base is A, the genotype is AA or AG, and the Holstein cow has high milk fat rate and milk protein rate; when the SNP molecular marker base is G, the genotype is GG, and the Holstein cow has high 305-day milk yield, and the milk quality is not significantly reduced. By applying the marker to genetic improvement of dairy cows, the frequency of the advantageous allele can be improved generation by generation by selecting individuals carrying the advantageous allele, so that the lactation performance of the dairy cow is effectively improved, the breeding efficiency is optimized, and the competitive advantage in the field of dairy cow breeding is enhanced. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Several mutations of the 1761th site of the Leptin gene in the application.

[0020] Figure 2 Prediction of the miRNA combined with the 1761th site of the Leptin gene in the application.

[0021] Figure 3 Insertion sequence of the psi-Lep1761G and psi-Lep1761A recombinant plasmid in the application.

[0022] Figure 4 Detection results of the luciferase reporter gene of the 1761th site of the Leptin gene in the application.

[0023] Figure 5 Detection results of the expression amount of the Leptin mRNA of different genotypes of the 1761th site of the Leptin gene in the application.

[0024] Figure 6 Experimental results of the bta-miR-502b targeted inhibition of the expression of the Leptin gene in the application. DETAILED DESCRIPTION

[0025] The technical solutions of the present application are further described below with reference to the drawings.

[0026] Example 1

[0027] In this embodiment, KASP (Kompetitive Allele-Specific PCR) technology is used for high-throughput genotyping of Holstein Leptin gene (NCBI database gene number NC_037333.1), as follows:

[0028] (1) Blood was collected from the tail vein of 1331 Holstein cows (from Hongze Ranch and Rizhao Fresh Pure Ranch), and total genomic DNA was extracted using a blood genomic extraction kit (Tiangen Biochemical Technology Co., Ltd. DP348). The DNA concentration and purity were measured, and the DNA mother liquor was diluted to 50 ng / μL and stored at -20℃ in a refrigerator.

[0029] (2) The extracted genomic DNA was used as a template for PCR reaction by specific PCR primers to amplify the 1761 site of the Leptin gene.

[0030] Table 1 Leptin KASP-PCR amplification primer information

[0031]

[0032]

[0033] The KASP-PCR reaction system is shown in Table 2 as follows:

[0034] Table 2 KASP-PCR reaction system

[0035]

[0036] The KASP-PCR reaction conditions are shown in Table 3 as follows:

[0037] Table 3 KASP-PCR reaction conditions

[0038]

[0039] (3) The PCR amplification product was sent to Genesky Biotechnology Co., Ltd. for sequencing, and the sequencing results were analyzed. ContigExpress software was used for statistical analysis of the sequencing results to obtain the genotype frequency distribution of each population and screen SNPs. The obtained data are recorded in Table 4.

[0040] Table 4 Leptin gene mutation site genotyping, allele frequency and significance of chi-square test

[0041]

[0042] The genotypes and alleles included in the leptin gene mutation sites are as follows.

[0043] Example 2

[0044] (1) The miRNA binding properties of the c.1761G>A site in the 3'UTR region of the Leptin gene were analyzed by the RNAhybird online prediction tool. The results showed that the G allele could form stable binding with bta-miR-502b, and the mfe value was -19.3 kcal / mol.

[0045] (2) According to the prediction results, bta-miR-502b was selected as the test miRNA, and the corresponding miRNA mimics were designed for cell level functional test. All miRNA mimics were synthesized by Suzhou Jimake Gene Biotechnology Co., Ltd. to ensure the synthesis purity and transfection efficiency. The mimics included negative control miRNA (miR-NC) and specific miRNA (such as bta-miR-502b), and the sequence information is shown in Table 6. All miRNA mimics were dissolved and stored at low temperature according to the manufacturer's instructions before use. In the test, they were transferred into 293T cells by liposome transfection method to verify their regulation effect on the expression of Leptin gene.

[0046] Table 6 miRNA mimics sequence

[0047]

[0048] (3) The mRNA sequence of Leptin gene was obtained through Ensembl database (https: / / asia.ensembl.org / index.html), and the 3'UTR fragment sequence of c.1761G>A was synthesized and cloned downstream of Renilla luciferase gene in psi-CHECK2 dual luciferase reporter vector to construct psi-Lep1761G and psi-Lep1761A recombinant plasmids. The insertion sequence is shown in Figure 3 . The vector construction was completed by Wuhan Jin Kai Rui Biological Engineering Co., Ltd.

[0049] (4) The miRNA binding specificity was verified by dual luciferase reporter gene experiment. 293T cells were inoculated in 12-well plates, and Lipofectamine 3000 was used to transfect the recombinant plasmids and miRNA mimics. The luciferase activity was detected 48 hours later. TM2000 transfection reagent, co-transfect recombinant plasmid (psi-Lep1761G or psi-Lep1761A) with miRNA mimic (bta-miR-502b mimic), set negative control (miR-NC) and blank vector control (psi-CHECK2). Use Dual-Luciferase Reporter Assay System to detect Firefly and Renilla luciferase activity, calculate relative activity ratio (F / R). Results are shown in Figure 4 psi-Lep1761G and bta-miR-502b co-transfection group was significantly lower than the miR-NC control group (P<0.01), while the relative fluorescence activity of psi-Lep1761A and bta-miR-502b co-transfection group had no significant difference (P>0.05), confirming that the c.1761G allele can specifically bind to bta-miR-502b and inhibit downstream gene expression, and the A allele loses this regulatory effect.

[0050] Example 3

[0051] In this embodiment, the expression amount of Leptin mRNA in individuals of different genotypes was detected by qRT-PCR technology.

[0052] (1) Select 3 adult lactating Holstein cows of different genotypes (GG and GA) at the c.1761G>A site, collect mammary tissue samples, extract total RNA using TRIzol reagent, and detect RNA integrity and purity (OD260 / OD280=1.8-2.0) by agarose gel electrophoresis and Nanodrop.

[0053] (2) Use GAPDH as an internal reference gene, design Leptin gene-specific primers, and the specific sequences are shown in Table 7.

[0054] Table 7 Primer sequences for target genes

[0055]

[0056] (3) Use Takara fluorescent quantitative kit, and configure the reaction solution according to Table 8.

[0057] Table 8 qRT-PCR system

[0058]

[0059] (4) Detect the expression of mRNA according to the procedure shown in Table 9.

[0060] Table 9 qRT-PCR procedure

[0061]

[0062] After the reaction, the product specificity was determined based on the melting curve, GAPDH was used as the internal reference gene for comparison, and the relative expression level of the target gene mRNA was calculated using the Ct method (2-ΔΔCt) according to the comparative cycle threshold method. Figure 5 As shown. Due to the extremely low frequency of AA homozygosity, only three samples each of the GG and GA genotypes were ultimately screened. qRT-PCR results showed that at the c.1761G>A locus, leptin expression levels in individuals with the GG genotype were significantly lower than those with the GA genotype (P<0.05), suggesting that the G allele is strongly inhibited by bta-miR-502b in mammary gland tissue. However, after the G to A mutation, this miRNA's regulatory ability is weakened, leading to a rebound in leptin expression. These results demonstrate that the c.1761G>A single nucleotide polymorphism affects leptin gene expression in mammary gland tissue of dairy cows.

[0063] Example 4

[0064] In this example, bta-miR-502b mimics (mimics) and negative control (miR-NC) were transfected into MAC-T bovine mammary epithelial cells (the cell line has the G allele at the c.1761G>A site). Total RNA was extracted 48 hours later and the expression of Leptin mRNA was detected by qRT-PCR. Figure 6 As shown. Figure 6 It can be seen that compared with the miR-NC group, the Leptin mRNA expression level in the bta-miR-502b transfection group was significantly downregulated (P<0.05), confirming that bta-miR-502b can target and inhibit Leptin gene expression, and this regulatory effect depends on the presence of the c.1761G allele.

[0065] Example 5

[0066] In this example, SPSS software multivariate analysis of variance and general linear model were used to perform association analysis on the genotype of the gene mutation site with five lactation traits, namely, milk fat percentage, milk protein, urea nitrogen, somatic cell score, and 305-day milk production. The model is as follows:

[0067] y kijl =μ+S i +parity j +DIM k +G l +e kijl

[0068] Among them, y kijlY represents the phenotypic value of the analyzed trait; μ is the population mean; S i are fixed effects for pasture, farrowing year and season; parity j is a fixed effect for parity (divided into three categories: 1st parity, 2nd parity and 3rd parity and above); DIM k is a fixed effect for lactation stage (divided into 10 stages of 30 days each); G l is a fixed effect for the lth genotype; e kijl is a random residual term.

[0069] The data obtained from the statistics of the effect of the gene mutation of the screened Leptin gene mutation site, i.e., various Leptin gene single nucleotide polymorphisms, on the lactation performance of Holstein cows is shown in Table 5.

[0070] Table 5 Effect of Leptin gene single nucleotide polymorphisms on the lactation performance of Holstein cows

[0071]

[0072] Note: The variance analysis uses the F test method. The F value in the results is a specific numerical value calculated by the F test formula. According to the value, the corresponding p value, i.e., the significance level (Sig.), can be obtained by table lookup or other methods. The significance marks indicate that *P < 0.05; **P < 0.01; the differences marked as a and b in the same column are significantly different at the P < 0.05 level.

[0073] As shown in Table 5, the Leptin gene single nucleotide polymorphisms have a significant effect on the lactation performance of Holstein cows. Among them, at the c.1761G>A site, the 305-day milk yield of the GG genotype (10630.49 ± 1925.23 kg) is 500.12 kg higher than that of the AA type (10130.37 ± 1064.03 kg) and 381.69 kg higher than that of the AG genotype (10248.8 ± 1878.84 kg), with a very significant difference (P < 0.01), and has no significant effect on milk fat rate, milk protein rate and somatic cell count, with no significant decrease in milk quality and stable milk quality. The urea nitrogen (14.45 ± 3.74 mg / dL) is significantly lower than that of other genotypes (P < 0.01), i.e., in the Leptin gene, different genotypes at the c.1761G>A site are accompanied by different lactation performances, and the GG type shows consistent advantages in milk yield (very significantly higher) and urea nitrogen (significantly lower), without affecting the milk fat rate and milk protein rate related milk quality, i.e., high milk yield and stable milk quality.

[0074] Based on the above results, SNP molecular marker can be designed, with the single nucleotide site at 1761 bp of cDNA sequence being G or A, which can be used as genetic marker for lactation performance of Holstein cow.

Claims

1. A SNP molecular marker for increasing milk production in Holstein cattle, characterized in that: The SNP molecular marker is located at the mutation site of the Leptin gene in the Holstein cattle genome. The gene number of the Leptin gene in the NCBI database is NC_037333.

1. The SNP molecular marker is located at the 1761st base of the Leptin gene cDNA sequence, and the mutant base is A or G.

2. The SNP molecular marker for increasing milk production in Holstein cattle according to claim 1, characterized in that: When the SNP molecular marker base is A, the genotype is AA or AG, and the milk fat rate and milk protein rate of the Holstein cow are high; when the SNP molecular marker base is G, the genotype is GG, and the 305-day milk production of the Holstein cow is high.

3. A primer pair for amplifying the SNP molecular marker according to any one of claims 1 or 2, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 1 to 3.

4. A reagent or kit comprising the primer pair according to claim 3.

5. The reagent or kit according to claim 4, characterized in that The kit also includes KASP MasterMix.

6. Use of the SNP molecular marker according to any one of claims 1 or 2, the primer pair according to claim 3, or the reagent or kit according to any one of claims 4 or 5 in detecting the lactation performance of Holstein cattle, or in Holstein cattle breeding.

7. The use according to claim 6, characterized in that Methods for testing the lactation performance of Holstein cattle or Holstein cattle breeding include: (1) Extracting genomic DNA from Holstein cattle blood as template DNA; (2) using a specific primer pair to perform PCR amplification on the genomic DNA of the Holstein cattle to be tested obtained in step (1) to obtain a PCR amplification product; (3) Purifying the PCR amplification product obtained in step (2) and performing genotyping detection. When the SNP molecular marker base is A, the genotype is AA or AG, and the milk fat rate and milk protein rate of the Holstein cow are high; when the SNP molecular marker base is G, the genotype is GG, and the 305-day milk production of the Holstein cow is high.

8. The use according to claim 7, characterized in that The PCR amplification system includes template DNA, upstream and downstream primers, KASP Master Mix and ddH2O.

9. The use according to claim 7, characterized in that The PCR amplification program was as follows: 95°C for 15 min; 94°C for 20 s; 61°C for 60 s, 10 cycles; 94°C for 20 s, 55°C for 60 s, 26 cycles, and 37°C for 1 min.

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