SNP molecular marker based on ILF3 gene and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker based on an ILF3 (Interleukin-3) gene and application thereof, and belongs to the technical field of genetic engineering, the molecular marker is positioned on a chromosome 7 of a Holstein cattle genome, and particularly relates to a mutation site of the ILF3 gene, and the number of the gene in NCBI (National Center of Biotechnology Information) is NC037334.1. The SNP site is located at the 12647 basic group of the ILF3 gene and shows T / C polymorphism. According to the invention, an efficient and accurate molecular marker assisted breeding technology is successfully constructed by verifying the association effect of the SNP molecular marker and the lactation character. The technology can be applied to genetic improvement of dairy cows, individuals with dominant alleles are selected, and the frequency of the alleles is increased generation by generation, so that the lactation performance of the dairy cows is effectively improved, and the core competitiveness of the dairy cows is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a SNP molecular marker based on an ILF3 gene and an application thereof. Background Art

[0002] With the large-scale and intensive development of modern dairy farming, improving the milk production performance of dairy cows has become one of the key goals to improve the efficiency of dairy cow breeding and the quality of dairy products. The lactation performance of dairy cows is affected by a combination of factors such as genetic factors, physiological factors and environmental factors. Among them, genetic factors play an important role in the lactation traits of dairy cows. Fat content (FC) and protein content (PC) in lactation traits are important indicators for measuring the quality of dairy cow milk, and have a direct impact on the nutritional value, processing quality and economic benefits of dairy products. These two indicators are usually closely related to the genetic background of dairy cows. Therefore, through genetic improvement, the fat and protein content can be effectively increased, thereby improving lactation performance and dairy product quality.

[0003] Traditional breeding methods mainly rely on phenotypic selection, but this method is greatly affected by environmental factors and the improvement progress is slow. With the development of molecular biology technology, genomics and genetic marker-assisted selection (MAS) provide new solutions for dairy cow breeding. Through in-depth research on the dairy cow genome, it has been proved that many genes or single nucleotide polymorphism (SNP) sites related to milk production traits can be used as effective genetic markers to assist breeding work. Therefore, the discovery and application of specific SNP molecular markers, especially those closely related to lactation traits, has become an effective way to improve the milk production performance of Holstein cows.

[0004] Interleukin EnhancerBindingFactor 3 (ILF3) is a gene that plays an important role in transcriptional regulation and gene expression regulation, and is widely involved in multiple physiological processes such as immune response, cell growth, and differentiation. Studies have shown that ILF3 has a promoting effect in a variety of tumors, such as the invasion and metastasis of gastric cancer, the formation and survival of lung cancer cells, and the proliferation and metastasis of breast cancer cells. In addition, ILF3 is closely related to lactation and mammary gland inflammation in dairy cows, and can indirectly affect mammary gland health and lactation function by regulating the response of immune cells in mammary gland tissue. In the physiological process of the mammary gland, ILF3 may promote the normal development of the mammary gland and milk synthesis by interacting with key transcription factors and signaling pathways. Further studies have shown that LncRNA can guide the localization of ILF3 protein in a dependent manner, thereby recruiting polysomes or translation initiation factors, accelerating the transcription and translation process, and enhancing the function of mammary cells. Therefore, the potential role of ILF3 in mammary gland development, milk synthesis, and mammary gland inflammation also provides important clues for further research. Summary of the invention

[0005] Technical problems to be solved: In view of the technical defects in the prior art, such as the fact that traditional breeding methods are greatly affected by environmental factors and the improvement progress is slow, a method based on SNP molecular markers of the ILF3 gene is disclosed. The study verifies the significant effect of the SNP molecular marker on lactation traits, and establishes an efficient and accurate molecular marker-assisted breeding technology. By applying the marker to the genetic improvement of dairy cows, it is possible to select individuals carrying dominant alleles and increase the frequency of dominant alleles from generation to generation, thereby effectively improving the lactation performance of dairy cows, optimizing breeding efficiency, and enhancing competitive advantage in the field of dairy cow breeding.

[0006] Technical solution:

[0007] A SNP molecular marker based on the ILF3 gene, wherein the SNP molecular marker is located on chromosome 7 of the Holstein cattle genome, specifically a mutation site of the ILF3 gene, and the gene number of the ILF3 gene in the NCBI database is NC_037334.1.

[0008] Further: the SNP molecular marker is located at the base position of the 12647th site of the ILF3 gene, and the site exhibits T / C polymorphism.

[0009] The primer pair used to amplify any of the above-mentioned SNP molecular markers based on the ILF3 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1: 5'-ATGCGTCCAGTGAGAAATTTTGTGAATG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-TTATCTGTATTGGTAGTTCATGCTGTGGTC-3'.

[0010] The present application also discloses a reagent or a kit containing the above-mentioned primer pair.

[0011] Use of any of the above-mentioned SNP molecular markers based on the ILF3 gene, the above-mentioned primer pair or the above-mentioned reagent or kit in improving the lactation performance of Holstein cattle.

[0012] Further: Among the genetic mutation types of the ILF3 gene, the lactation performance of CC genotype individuals is better than that of CT and TT types.

[0013] Furthermore: The daily milk production and 305-day milk production of CC genotype individuals were significantly higher than those of CT and TT genotypes, the fat percentage of CT genotype individuals was significantly higher than that of CC and TT genotypes, and the somatic cell score of CC and CT genotype individuals was significantly higher than that of TT type.

[0014] Application of any of the above-mentioned SNP molecular markers based on the ILF3 gene, the above-mentioned primer pair or the above-mentioned reagent or kit in breeding high-quality and high-yield Holstein cattle.

[0015] Beneficial effects:

[0016] 1. Through systematic experimental research, the present invention finally determined the SNP molecular marker related to the lactation performance of Holstein cattle. The marker is located on chromosome 7 of the Holstein cattle genome and involves the mutation of the ILF3 gene;

[0017] 2. The SNP molecular marker is located at the 12647th site of the ILF3 gene, which has a T / C polymorphism. The present invention uses the SNP marker to design specific primers, detects the site by PCR amplification, and uses Sequenom MassARRAY technology to perform SNP-based genotyping on 992 Holstein cows. The correlation between the ILF3-g.12647T>C site genotype and lactation traits is analyzed by the least squares method. The present invention reveals the pleiotropic effect of the ILF3 gene in Holstein cows in lactation traits, and finds different dominant genotypes for the ILF3-g.12647T>C site, which provides a theoretical basis for subsequent molecular genetic research on the lactation performance of Holstein cows. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the three genotype diagrams of the ILF3-g.12647T>C site in this application. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] The technical means used in the following examples, unless otherwise specified, are all conventional means well known to those skilled in the art, and the experimental methods without specifying specific conditions are all conventional methods in the art.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] The Holstein ILF3 gene source used in the following examples is a sample statistics from six pastures in Jiangsu Province, and the pastures statistically include: Huaxia Pasture, Xuyi Pasture, Sihong Pasture, Xinyi Pasture, Shenfeng Pasture, Yonghao Pasture, and the statistical data include sampling date, calving date, calving interval, lactation days, grouping number, milk yield, milk fat rate, protein rate, fat-egg ratio, somatic cell number, somatic cell score, urea nitrogen, milk loss, milk price difference, economic loss, correction milk, persistence, WHI, foremilk volume, precursor cell, precursor cell score, foremilk loss, peak milk, peak day, 305 milk volume, total milk volume, total milk fat, total protein, and adult equivalent. The data obtained are processed as shown in Table 3.

[0023] Example 1

[0024] The single nucleotide polymorphism of ILF3 gene in Holstein cattle was screened as follows:

[0025] (1) Blood was collected from the caudal vein of 992 Holstein cattle, and total genomic DNA was extracted using a blood genome extraction kit. The DNA concentration and purity were measured, and the DNA mother solution was diluted to 50 ng / μL and stored in a -20°C refrigerator.

[0026] (2) Using the extracted genomic DNA as a template, a conventional PCR reaction was performed using PCR primers. The amplification primers used are as follows:

[0027] The nucleotide sequence of the upstream primer ILF3-F is shown in SEQ ID NO. 1: 5′-ATGCGTCCAGTGAGAAATTTTGTGAATG-3′;

[0028] The nucleotide sequence of the downstream primer ILF3-R is shown in SEQ ID NO.2: 5'-TTATCTGTATTGGTAGTTCATGCTGTGGTC-3'.

[0029] The PCR reaction system is shown in Table 1 below:

[0030] Table 1 PCR reaction system

[0031]

[0032] The PCR reaction conditions are shown in Table 2 below:

[0033] Table 2 PCR reaction conditions

[0034]

[0035] (3) The PCR amplification products were sent to Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results were analyzed using ContigExpress software. The ILF3-g.12647T>C site was screened out through analysis. This site is located at position 12647 of the ILF3 gene on chromosome 7 of the Holstein cattle genome. The mutant base is T or C. Figure 1 As shown. According to the different base compositions of this site, three genotypes are formed: CC type, CT type and TT type. The gene number of ILF3 gene in NCBI database is NC_037334.1.

[0036] Example 2

[0037] In this example, SPSS software multi-factor variance analysis and general linear model method were used to perform association analysis on the genotype of the gene mutation site and the four lactation traits of daily milk production, milk fat percentage, somatic cell score and 305-day milk production. The model is as follows:

[0038] Y ijklmno =μ+CS i +MS j +P k +D l +G m +F n +e ijklmno

[0039] Among them, Y ijklmno is the observed value of lactation performance, μ is the group mean, CS i is the fixed effect of calving season, MS j To measure the fixed effect of season, P k is the fixed effect of parity, D l is the fixed effect of lactation days, G m is the fixed effect of genotype, F n is the field effect, e ijklmno Random variation.

[0040] The effects of the gene mutation type of the ILF3-g.12647T>C site screened, i.e., multiple ILF3 gene single nucleotide polymorphisms on the lactation performance of Holstein cows, were statistically analyzed, and the obtained data are recorded in the following Table 3.

[0041] Table 3 Effects of ILF3 gene single nucleotide polymorphism on lactation performance of Holstein cattle

[0042]

[0043]

[0044] Notes to Table 3: Data in the same column with different lowercase letters indicate significant differences (P < 0.05).

[0045] The ILF3-g.12647T>C locus significantly affected daily milk yield, milk fat percentage, somatic cell score and 305-day milk yield (P<0.05). Among them, individuals with CC genotype had significantly higher daily milk yield and 305-day milk yield than those with CT and TT genotypes (P<0.05); the milk fat percentage of CT genotype individuals was significantly higher than that of CC genotype (P<0.05), but there was no significant difference with TT genotype (P>0.05); in addition, the somatic cell score of CC and CT genotypes was significantly higher than that of TT genotype (P<0.05). These results indicate that different genotypes at the 12647th locus of ILF3 gene are closely related to lactation performance, among which CC genotype shows better lactation performance.

[0046] Based on the above research results, a SNP molecular marker can be designed, whose nucleotide sequence has a single nucleotide site of T or C at the 12647 bp position, as a genetic marker for the lactation performance of Holstein cattle.

[0047] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A SNP molecular marker based on the ILF3 gene, characterized in that: The SNP molecular marker is located on chromosome 7 of the Holstein cattle genome, specifically the mutation site of the ILF3 gene. The gene number of the ILF3 gene in the NCBI database is NC_037334.

1.

2. The SNP molecular marker based on the ILF3 gene according to claim 1, characterized in that: The SNP molecular marker is located at the base position of the 12647th site of the ILF3 gene, and the site exhibits T / C polymorphism.

3. A primer pair for amplifying the SNP molecular marker based on the ILF3 gene according to claim 1, characterized in that: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1: 5'-ATGCGTCCAGTGAGAAATTTTGTGAATG-3', and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2: 5'-TTATCTGTATTGGTAGTTCATGCTGTGGTC-3'.

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

5. Use of the SNP molecular marker based on the ILF3 gene as claimed in claim 1 or 2, the primer pair as claimed in claim 3, or the reagent or kit as claimed in claim 4 in improving the lactation performance of Holstein cattle.

6. The use according to claim 5, characterized in that: Among the genetic mutation types of the ILF3 gene, the lactation performance of individuals with the CC genotype is better than that of individuals with the CT and TT genotypes.

7. The use according to claim 6, characterized in that: The daily milk production and 305-day milk production of CC genotype individuals were significantly higher than those of CT and TT genotypes, the fat percentage of CT genotype individuals was significantly higher than those of CC and TT genotypes, and the somatic cell score of CC and CT genotype individuals was significantly higher than that of TT genotype.

8. Use of the SNP molecular marker based on the ILF3 gene as claimed in claim 1 or 2, the primer pair as claimed in claim 3, or the reagent or kit as claimed in claim 4 in breeding high-quality and high-yield Holstein cattle.

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