SNP molecular marker based on ilf3 gene and application thereof

Abstract

The application discloses a SNP molecular marker based on an ILF3 gene and application thereof, and belongs to the technical field of genetic engineering, wherein the molecular marker is located on the 7th chromosome of a Holstein genome, specifically a mutation site of an ILF3 gene, and the gene is numbered as NC_037334.1 in NCBI. The SNP site is located at the 12635th base of the ILF3 gene, and shows T / C polymorphism. The application successfully constructs a high-efficiency and accurate molecular marker assisted breeding technology by verifying the correlation effect of the SNP molecular marker and a lactation performance. The technology can be applied to genetic improvement of dairy cows, and the lactation performance of the dairy cows can be effectively improved, and the core competitiveness of the dairy cows can be enhanced by selecting individuals with advantageous alleles and increasing the frequency of the alleles from generation to generation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an SNP molecular marker based on the ILF3 gene and its application. Background Technology

[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 objectives for enhancing dairy cow breeding efficiency and dairy product quality. The milk production performance of dairy cows is influenced by a combination of factors, including genetic, physiological, and environmental factors, among which genetic factors play a crucial role. Fat content (FC) and protein content (PC) are important indicators for measuring the quality of dairy cow milk, directly impacting 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, genetic improvement can effectively increase the fat and protein content, thereby improving milk production performance and dairy product quality.

[0003] Traditional breeding methods primarily rely on phenotypic selection, but this approach is highly susceptible to environmental interference and progresses slowly. With the development of molecular biology techniques, genomics and marker-assisted selection (MAS) have provided new solutions for dairy cattle breeding. In-depth research into the dairy cattle genome has demonstrated that many genes or single nucleotide polymorphism (SNP) sites associated with milk production traits can serve as effective genetic markers to assist breeding efforts. Therefore, discovering and applying specific SNP molecular markers, especially those closely related to lactation traits, has become an effective way to improve the milk production performance of Holstein cattle.

[0004] Interleukin Enhancer Binding Factor 3 (ILF3) is a gene that plays a crucial role in transcriptional regulation and gene expression regulation, and is widely involved in multiple physiological processes such as immune responses, cell growth, and differentiation. Studies have shown that ILF3 promotes various 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. Furthermore, 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 tissue. In the physiological processes of the mammary gland, ILF3 may promote normal mammary gland development and milk synthesis by interacting with key transcription factors and signaling pathways. Further research has shown that lncRNA can dependently guide the localization of ILF3 protein, thereby recruiting polyribosomes or translation initiation factors, accelerating transcription and translation processes, and enhancing mammary cell function. Therefore, the potential role of ILF3 in mammary gland development, milk synthesis, and mammary gland inflammation provides important clues for further research. Summary of the Invention

[0005] Technical problems solved: Addressing the shortcomings of existing traditional breeding methods, such as significant susceptibility to environmental factors and slow improvement progress, this paper discloses a method based on the ILF3 gene SNP molecular marker. Research has verified the significant role of this SNP molecular marker in lactation traits and established an efficient and precise molecular marker-assisted breeding technology. By applying this marker to dairy cow genetic improvement, the frequency of dominant alleles can be increased generation by generation by selecting individuals carrying dominant alleles, thereby effectively improving the lactation performance of dairy cows, optimizing breeding efficiency, and enhancing competitive advantage in the dairy cow breeding field.

[0006] Technical solution: A SNP molecular marker based on the ILF3 gene, wherein the SNP molecular marker is located on chromosome 7 of the Holstein bovine genome, specifically at the mutation site of the ILF3 gene, and the gene number of the ILF3 gene in the NCBI database is NC_037334.1.

[0007] Furthermore, the SNP molecular marker is located at the 12635th base position of the ILF3 gene, which exhibits T / C polymorphism.

[0008] The primer pairs used to amplify any of the above-mentioned SNP molecular markers based on the ILF3 gene have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.1: 5'-ATGCGTCCAGTGAGAAATTTTGTGAATG-3', and downstream primer as shown in SEQ ID NO.2: 5'-TTATCTGTATTGGTAGTTCATGCTGTGGTC-3'.

[0009] This application also discloses a reagent or kit containing the primer pairs described above.

[0010] The application of any of the above-mentioned SNP molecular markers based on the ILF3 gene, the above-mentioned primer pairs, or the above-mentioned reagents or kits in improving the lactation performance of Holstein cattle.

[0011] Furthermore, among the ILF3 gene mutants, individuals with the CC genotype have better lactation performance than those with the CT and TT genotypes.

[0012] Furthermore, individuals with the CC genotype had significantly higher daily milk production and 305-day milk production than those with the CT and TT genotypes. Individuals with the CT genotype had significantly higher lipid percentage than those with the CC and TT genotypes. Individuals with the CC and CT genotypes had significantly higher somatic cell scores than those with the TT genotype.

[0013] The application of any of the above-mentioned SNP molecular markers based on the ILF3 gene, the above-mentioned primer pairs, or the above-mentioned reagents or kits in the breeding of high-quality and high-yielding Holstein cattle.

[0014] Beneficial effects: 1. Through systematic experimental research, this invention finally identified the SNP molecular marker associated with lactation performance in Holstein cattle. This marker is located on chromosome 7 of the Holstein cattle genome and involves mutations in the ILF3 gene; 2. The SNP molecular marker is located at position 12635 of the ILF3 gene, which exhibits T / C polymorphism. This invention utilizes this SNP marker to design specific primers, amplifies and detects this position by PCR, and simultaneously uses Sequenom MassARRAY technology to perform SNP-based genotyping on 992 Holstein cattle; the correlation between the ILF3-g.12635 T>C genotype and lactation traits is analyzed using the least squares method; this invention reveals the pleiotropic effect of the Holstein cattle ILF3 gene in lactation traits, and identifies different dominant genotypes at the ILF3-g.12635 T>C site, providing a theoretical basis for subsequent molecular genetic studies on the lactation performance of Holstein dairy cattle. Attached Figure Description

[0015] Figure 1 This is a diagram of three genotypes at the ILF3-g. 12635 T>C locus in this application. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0017] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] The Holstein ILF3 gene used in the following examples was obtained from samples collected from six ranches in Jiangsu Province: Huaxia Ranch, Xuyi Ranch, Sihong Ranch, Xinyi Ranch, Shenfeng Ranch, and Yonghao Ranch. The collected data included sampling date, calving date, calving interval, lactation days, group number, milk yield, milk fat percentage, protein percentage, fat-to-protein ratio, somatic cell count, somatic cell fraction, blood urea nitrogen, milk loss, milk quality difference, economic loss, corrected milk, sustainability, WHI, foremilk yield, precursor cells, precursor cell fraction, foremilk loss, peak milk yield, peak day, 305 milk yield, total milk yield, total milk fat, total protein, and adult equivalent. The processed data are shown in Table 3.

[0020] Example 1: Screening for single nucleotide polymorphisms in the Holstein bovine ILF3 gene, as detailed below:

[0021] (1) Blood was collected from the tail vein of 992 Holstein cattle. Total genomic DNA was extracted using a blood genomic extraction kit. The DNA concentration and purity were measured. The DNA stock solution was aliquoted and diluted to 50 ng / μL and stored at -20℃.

[0022] (2) Using the extracted genomic DNA as a template, a conventional PCR reaction was performed using PCR primers. The amplification primers used are shown below:

[0023] The nucleotide sequence of the upstream primer ILF3-F is shown in SEQ ID NO.1: 5'-ATGCGTCCAGTGAGAAATTTTGTGAATG-3';

[0024] The nucleotide sequence of the downstream primer ILF3-R is shown in SEQ ID NO.2: 5'-TTATCTGTATTGGTAGTTCATGCTGTGGTC-3'.

[0025] The PCR reaction system is shown in Table 1 below:

[0026] Table 1 PCR reaction system .

[0027] The PCR reaction conditions are shown in Table 2 below:

[0028] Table 2 PCR reaction conditions .

[0029] (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.12635 T>C site was identified through analysis. This site is located at position 12635 of the ILF3 gene on chromosome 7 of the Holstein bovine genome, with a mutated base of T or C. Figure 1 As shown. Based on the different base compositions at this site, three genotypes are formed: CC, CT, and TT. The gene ID of the ILF3 gene in the NCBI database is NC_037334.1.

[0030] Example 2: In this example, SPSS software was used to perform multivariate analysis of variance and a general linear model to analyze the association between the genotype of the gene mutation site and four lactation traits: daily milk yield, milk fat percentage, somatic cell score, and 305-day milk yield. The model is as follows: Among them, Y ijklmno CS represents the observed value of lactation performance, μ is the population mean, and CS is the mean. i For the fixed effect of calving season, MS j To determine the seasonal fixed effect, P k For the fixed effect of parity, D l For the fixed effect of lactation days, G m For the genotype fixation effect, F n For field effect, e ijklmno It is random and uneven.

[0031] The effects of the ILF3-g.12635 T>C site gene mutations (i.e., multiple ILF3 gene single nucleotide polymorphisms) on the lactation performance of Holstein cattle were statistically analyzed, and the data obtained are recorded in Table 3 below.

[0032] Table 3. Effects of ILF3 gene single nucleotide polymorphism on lactation performance in Holstein cattle. .

[0033] Table 3 Note: Different lowercase letters in the superscript of data in the same column indicate significant differences. P <0.05).

[0034] The ILF3-g.12635 T>C locus significantly affected daily milk yield, milk fat percentage, somatic cell score, and 305-day milk yield (P < 0.05). Specifically, individuals with the CC genotype had significantly higher daily and 305-day milk yields than those with the CT and TT genotypes (P < 0.05); individuals with the CT genotype had a significantly higher milk fat percentage than those with the CC genotype (P < 0.05), but no significant difference compared to the TT genotype (P > 0.05); furthermore, the somatic cell scores of the CC and CT genotypes were significantly higher than those of the TT genotype (P < 0.05). These results indicate that different genotypes at ILF3 locus 12635 are closely related to lactation performance, with the CC genotype exhibiting better lactation performance.

[0035] Based on the above research results, an SNP molecular marker can be designed, whose nucleotide sequence has a single nucleotide site of T or C at 12635 bp, to serve as a genetic marker for the lactation performance of Holstein cattle.

[0036] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of an SNP molecular marker based on the ILF3 gene in screening for lactation performance in Holstein cattle, characterized in that, The lactation performance is defined as daily milk yield, milk fat percentage, somatic cell score, and milk yield over 305 days. The SNP molecular marker is located at the 12635th base position of the ILF3 gene, which exhibits T / C polymorphism. The gene number of the ILF3 gene in the NCBI database is NC_037334.

1.

2. The application according to claim 1, characterized in that: Individuals with the CC genotype had significantly higher daily milk production and 305-day milk production than those with the CT and TT genotypes. Individuals with the CT genotype had significantly higher milk fat percentage than those with the CC and TT genotypes. Individuals with the CC and CT genotypes had significantly higher somatic cell scores than those with the TT genotype.

3. The application of a primer pair for detecting SNP molecular markers, or a reagent or kit containing such primer pair, in the breeding of Holstein cattle with high-quality, high-yielding lactation performance, characterized in that... The lactation performance is defined as daily milk yield, milk fat percentage, somatic cell score, and milk yield over 305 days. The SNP molecular marker is located at the 12635th base position of the ILF3 gene, which exhibits T / C polymorphism. The gene number of the ILF3 gene in the NCBI database is NC_037334.1.

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