Application of chicken miRNA-3528 gene SNP molecular marker in chicken economic character improvement breeding and chicken blood biochemical index evaluation

Abstract

The invention relates to application of a chicken miRNA-3528 gene SNP molecular marker in chicken economic character improvement breeding and chicken blood biochemical index evaluation, and belongs to the technical field of biological breeding. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the 12th basic group from the 5'end is A or G; the economic traits are carcass traits and / or growth traits and / or meat quality traits; the carcass traits comprise full eviscerating weight; the growth traits comprise birth weight, 2-week weight, 4-week weight and 6-week weight; the meat quality characters comprise sebum rate and pectoral muscle density. By screening and detecting the genotype of the SNP molecular marker of the chicken miRNA-3528 gene on the DNA level and selecting GG genotype cocks and hens for breeding, the accuracy of chicken body size character selection can be improved, the breeding process of new broiler varieties is accelerated, the production performance of chickens is effectively improved, and remarkable economic benefits are obtained.

Description

Technical Field

[0001] The invention relates to application of a chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding and chicken blood biochemical index evaluation, belonging to the technical field of biological breeding. Background Art

[0002] With the rapid development of the social economy and the improvement of people's living standards, the overall market demand for meat quality has shifted from quantitative requirements to the pursuit of quality and flavor. Growth traits, body size traits, meat quality traits, and carcass traits are the economic traits of greatest concern to the poultry industry today. Most economic traits are quantitative traits, primarily determined by a large number of genes with minor effects. These genes have additive, dominant, and epistatic effects. The genetic basis of complex traits is determined by the genetic and environmental factors that influence the phenotype, as well as the interactions between them. Currently, high-quality local chickens in China generally suffer from slow growth, which affects economic benefits and limits the further development of the chicken farming industry. Therefore, for many years, researchers have attempted to identify candidate genes associated with relevant economic traits in chickens through extensive experiments, providing auxiliary marker selection for molecular selection and breeding of poultry.

[0003] Molecular markers are genetic markers based on nucleotide sequence variation within the genetic material of individuals and are a direct reflection of genetic diversity at the DNA level. Molecular marker-assisted breeding utilizes the characteristic that molecular markers are closely linked to the genes that determine target traits. By detecting molecular markers, the presence of the target gene can be detected, achieving the purpose of selecting the target trait. It has the advantages of being fast, accurate, and not affected by environmental conditions. Modern breeding technology using molecular markers is an important way to accelerate the selection of improved varieties and improve the genetic quality of the population. The application of molecular marker breeding first involves screening and detecting genetic markers closely related to economic traits of chickens at the DNA level, followed by establishing rapid detection methods for their genetic polymorphisms, and then implementing genetic marker-assisted selection and achieving early diagnosis and selection.

[0004] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphisms caused by variations in a single nucleotide at the genomic level. SNPs can affect gene function at the DNA, RNA, and protein levels and are the most common type of heritable variation in humans, accounting for over 90% of known polymorphisms. Analyzing the functions of SNPs that influence gene products is of paramount importance. Association analysis between SNP diversity and economic traits has long been a hot topic in livestock genetics research. With the discovery and in-depth study of miRNAs, researchers have discovered that miRNAs not only participate in the molecular regulation of complex animal phenotypes, but also that SNP variations can lead to abnormal miRNA function, thereby causing variation in biological phenotypic traits. The mechanisms of miRNA-related polymorphisms include insertions, deletions, translocations, amplifications, and base substitutions. MiRNA polymorphism is a key factor influencing miRNA regulatory function. Abnormal expression of a single miRNA can affect the expression of hundreds of target genes. Reduced expression of certain key proteins can lead to abnormal physiological function and disease. Studies have found that polymorphisms in miRNA primary products, precursors, and mature forms can potentially affect the expression and pathways of hundreds of genes, thereby widely affecting miRNA function. Currently, there is little research on whether SNP molecular markers present in miRNAs can affect the economic traits of poultry. Summary of the Invention

[0005] The first purpose of the present invention is to provide an application of chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding, and to provide a rapid and accurate molecular breeding method based on chicken miRNA-3528 gene single nucleotide polymorphism.

[0006] The second object of the present invention is to provide an application of a chicken miRNA-3528 gene SNP molecular marker in evaluating chicken blood biochemical indicators.

[0007] In order to achieve the above-mentioned object, the technical solution of the present invention for the application of a chicken miRNA-3528 gene SNP molecular marker in the improvement and breeding of economic traits of chickens is as follows:

[0008] A chicken miRNA-3528 gene SNP molecular marker is used in chicken economic trait improvement breeding, characterized in that: the nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the 12th base from the 5' end is A or G; the economic trait is a carcass trait and / or a growth trait and / or a meat quality trait; the carcass trait includes eviscerated weight; the growth trait includes birth weight, 2-week body weight, 4-week body weight, and 6-week body weight; and the meat quality trait includes skin fat percentage and breast muscle density.

[0009] The beneficial effect of the above technical solution is that the application of the chicken miRNA-3528 gene SNP molecular marker in the improvement and breeding of chicken economic traits is a pioneering invention. The present invention first screens and verifies the miRNA SNP site, uses the chicken whole genome DNA as a template, designs primers to amplify the target fragment including the miRNA-3528 precursor, and after purification and sequencing, it is known that there is a base polymorphism of A or G at the 12th position of the chicken miRNA-3528 gene; then, specific primers are designed to amplify the gene fragment containing the polymorphic site, and the MassARRAY system matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology combined with a single primer extension reaction is used to perform miRNA SNP analysis on a large number of samples from the F2 generation resource group of Gushi chicken and Anka chicken. Genotyping of the single SNP locus A-3528 (rs14098602A / G) and association analysis with weight traits in the F2 generation resource population revealed a significant association between the single nucleotide polymorphism (SNP) at the miRNA-3528 gene and weight in chickens. The GG genotype significantly increased eviscerated weight, birth weight, two-week-old weight, and four-week-old weight (p<0.05). Using this genotype as a marker for weight-adjusted chickens and molecular genetic breeding could rapidly establish flocks with superior genetic resources. This detection method is accurate, reliable, rapid, sensitive, and cost-effective, making it suitable for use in chicken selection and molecular breeding, playing a crucial role in improving weight-adjusted chickens.

[0010] As a further improvement, the genotype of the SNP molecular marker is detected, and individuals with the SNP molecular marker having the GG genotype are selected.

[0011] As a further improvement, the detection includes the following steps: using the DNA of the chicken to be tested as a template, performing PCR amplification, adding a single base extension primer to perform a single base extension reaction, and performing flight mass spectrometry detection on the reaction product.

[0012] As a further improvement, the nucleotide sequences of the PCR primers are shown in SEQ ID NOs. 2-3.

[0013] As a further improvement, the nucleotide sequence of the single-base extension primer is shown in SEQ ID NO.4.

[0014] As a further improvement, the detection further includes a SAP enzyme digestion reaction after PCR amplification.

[0015] In order to achieve the above-mentioned object, the technical solution of the present invention for the application of a chicken miRNA-3528 gene SNP molecular marker in the evaluation of chicken blood biochemical indicators is as follows:

[0016] A chicken miRNA-3528 gene SNP molecular marker is used to evaluate chicken blood biochemical indicators. The application is for non-disease diagnosis purposes. The nucleotide sequence of the SNP molecular marker is as shown in SEQ ID NO.1, and the 12th base from the 5' end is A or G. The blood biochemical indicators include total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase.

[0017] The beneficial effect of the above technical solution is that: through a large number of experiments, the present invention found that the chicken miRNA-3528 gene SNP single nucleotide polymorphism is significantly correlated with protein, albumin, globulin, cholinesterase, and lactate dehydrogenase in chicken blood biochemical indicators, which can be used as a molecular marker to assist breeding, and also lay the foundation for precise nutritional regulation, health management optimization, and improvement of meat quality and yield.

[0018] As a further improvement, the genotype of the SNP molecular marker is detected. When the genotype of the chicken to be tested is GG, the chicken to be tested is an individual with low total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase; when the genotype of the chicken to be tested is AA, the chicken to be tested is an individual with high total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase.

[0019] As a further improvement, individuals with the SNP molecular marker GG genotype were selected.

[0020] Specifically, the application of chicken blood biochemical indicators in comprehensive production is as follows:

[0021] ① Molecular marker-assisted breeding: By screening individuals with the GG genotype, a flock of chickens with high growth performance and high feed efficiency can be quickly established, shortening the breeding cycle.

[0022] ②Precise nutritional regulation: Optimize feed formula (such as increasing energy density and balancing amino acid ratios) based on the metabolic characteristics of GG genotype chickens to maximize growth potential.

[0023] ③ Optimization of health management: Taking advantage of the characteristics of increased high-density lipoprotein HDL and reduced immune stress, reduce the use of antibiotics and develop a green farming model.

[0024] ④ Improvement of meat quality and yield: By regulating the activity of lactate dehydrogenase LDH and creatine phosphokinase CK, muscle quality and meat yield can be improved to meet market demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SNP sequencing map of miR-3528 in Example 1 of the present invention;

[0026] Figure 2The figure shows the mass spectra of SNPs at different sites of miR-3528 in Example 1 of the present invention (the upper figure represents the AA genotype, the middle figure represents the AG genotype, and the lower figure represents the GG genotype);

[0027] Figure 3 This is a cumulative graph of individuals with different genotypes of miR-3528 in Example 1 of the present invention (where yellow, green, and blue represent individuals with different miRNA genotypes, respectively);

[0028] Figure 4 This is a diagram of the miRNA secondary structure formed by SNPs at different sites of miR-3528 in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] Chicken is an important economic poultry and also an important model organism in scientific research. Growth traits are the main indicators for evaluating chicken production performance, while body size traits and carcass traits can reflect the growth rate and meat production performance of chickens. These three traits are all very important economic traits. Therefore, studying the growth traits, body size traits and carcass traits of chickens has important economic value. How to improve the economic traits of local livestock and poultry breeds without changing the germplasm characteristics and maintain the sustainable development of local livestock and poultry is an urgent problem to be solved. Based on this, the present invention provides an application of a chicken miRNA-3528 gene SNP molecular marker in the improvement and breeding of chicken economic traits.

[0030] By screening and detecting the genotype of the chicken miRNA-3528 gene SNP molecular marker at the DNA level and selecting male and female chickens with the GG genotype for breeding, the accuracy of chicken body size trait selection can be increased, the breeding process of new broiler chicken varieties can be accelerated, the production performance of chickens can be effectively improved, and significant economic benefits can be obtained.

[0031] The present invention will be further described below in conjunction with specific embodiments, but the scope of protection of the present invention is not limited thereto; however, these embodiments are merely illustrative and do not constitute any limitation on the scope of the present invention. The details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements fall within the scope of protection of the present invention. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0032] Example 1 Application of a Chicken miRNA-3528 Gene SNP Molecular Marker in Chicken Economic Trait Improvement and Breeding

[0033] The application of the chicken miRNA-3528 gene SNP molecular marker in the chicken economic trait improvement breeding of this embodiment is specifically implemented as follows:

[0034] 1. Detection of Single Nucleotide Polymorphisms in Chicken miRNA-3528 Gene

[0035] 1. Animal materials:

[0036] Gushi chickens are a premium local breed of yellow chicken in my country. They were developed naturally through long periods of closed breeding in Gushi County, under exceptional ecological and breeding conditions. Gushi chickens are dual-purpose chickens with excellent traits: first, they tolerate roughage and are resistant to disease, making them suitable for free-range grazing. Second, they have tender, flavorful meat, rich, nutritious broth, and a strong tonic effect. Third, their hens produce large, densely packed eggs with thick, dark-yellow egg whites and thick shells, making them durable for storage and transportation.

[0037] Anka broiler is one of the best broiler chicken breeds in the world today. It is also the fastest growing red and yellow feather broiler in China. It has the characteristics of strong adaptability, stress resistance, fast growth rate and high feed conversion rate.

[0038] 2. Animal breeding:

[0039] The Gushi-Anka stock population was established using an F-2 distant half-sib design. Seven families were established: four orthocross lines (Anka♂×Gushi♀) and three reciprocal lines (Gushi♂×Anka♀). The F0 generation was constructed by selecting breeders from both the dual-purpose Gushi and broiler Anka lines, mating at a ratio of 1:6 between males and females. The selected males and females were required to exhibit the characteristics of the breed, including high egg production, medium body weight, and pure pedigrees, to ensure heterozygosity at all loci in the F1 generation. One male was selected from the F1 progeny of each family and mated with hens from other families at a ratio of 1:9 between males and females to produce the F2 generation. Hens were required to be unrelated to the mating males and females. F1 breeder hens were also dispersed throughout the families as much as possible. Individuals with diverse appearance and heterozygosity were selected to ensure a high degree of separation of traits in the F2 generation.

[0040] 3. Feeding method:

[0041] The chickens were raised at a university experimental chicken farm in Henan Province, and subsequent experiments were conducted at the university's poultry genetic improvement laboratory. The birds were mixed across families, caged, and provided with free access to feed and water. Blood samples were collected from the birds until 12 weeks of age. Body measurements and growth traits were recorded weekly from 0 to 12 weeks of age.

[0042] 4. Extraction of genomic DNA and construction of mixed pool:

[0043] Using F2 chickens from the Gushi-Anka stock flock as the test material, 5 mL of blood was drawn from the jugular vein of each chicken, anticoagulated with EDTA (10 mmol / L Tris-Cl, 0.2 mmol / L EDTA, pH 8.0), and stored at -80°C. Genomic DNA was extracted from the preserved blood samples of F2 stock flock individuals using the phenol-chloroform method. DNA samples were first analyzed by 1% agarose gel electrophoresis, followed by determination of purity and concentration using a NanoDrop 2000 microspectrometer. DNA samples that failed the test were re-extracted. DNA samples from 100 randomly selected individuals were then pooled for screening.

[0044] 5. Prediction and classification annotation of SNP sites:

[0045] By querying miRNA-SNiPer( http: / / www.integratomics-time.com / miRNA-SNiPer / ) and dbSNP( http: / / www.ncbi.nlm.nih.gov / snp ) database, combined with http: / / www.mirbase.org / The screened SNPs were classified and annotated using the 20.0 database. The corresponding miRNA precursor sequences were obtained from Genebank, and the SNPs' locations on the miRNAs were determined using Blast. The miRNA locations on the genome were also analyzed using NCBI. SNPs were classified as located in the precursor, mature, and germplasm regions based on their location on the miRNAs. They were also classified as located in host genes or non-host genes based on their location on the genes. Annotation criteria included the SNP's location in the precursor, mature, and other miRNAs, and whether there was a host gene.

[0046] 6. SNP screening primers:

[0047] The target miRNA precursor sequence, including the preceding and following 500 bp, was downloaded from NCBI and four primer pairs were designed. A mixed DNA pool was constructed from 100 individuals randomly selected from the F2 generation resource population. PCR amplification was performed using the aforementioned primer pairs using the pooled DNA as a template. The PCR reaction system consisted of: 25.0 μL total; 12.0 μL 2× Taq PCR Master Mix (MBI), 1.0 μL forward primer (10 pmol / L), 1.0 μL reverse primer (10 pmol / L), 0.5 μL DNA template (100 ng / μL), and 10.5 μL ddH2O.

[0048] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60.2°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min.

[0049] PCR products were directly sequenced (Shanghai Bioengineering), and SNPs were determined based on whether there were overlapping peaks in the sequencing.

[0050] Primer pair 1:

[0051] Upstream primer: 5'-CAGTGTTGTGTACGTTGTCTGCTC-3' (as shown in SEQ ID NO.5),

[0052] Downstream primer: 5'-GTCAAGAAGTTGCTGACAGCATTG-3' (as shown in SEQ ID NO. 6);

[0053] Primer pair 2:

[0054] Upstream primer: 5'-CTTGTGAAGTTGCTCAGAGATTAAGTCAG-3' (as shown in SEQ ID NO.7),

[0055] Downstream primer: 5'-CAGCTGGCCTCACAGGATTTACTATG-3' (as shown in SEQ ID NO. 8);

[0056] Primer pair 3:

[0057] Upstream primer: 5'-TGTGACAAAGCTGCTTATAACTGCTTATAG-3' (as shown in SEQ ID NO.9),

[0058] Downstream primer: 5'-TGTTTTTAATCTCGCCATTATACGCGCTGC-3' (as shown in SEQ ID NO. 10);

[0059] Primer pair 4:

[0060] Upstream primer: 5'-GAGAGATCTTGGATTACAAG-3' (as shown in SEQ ID NO.11),

[0061] Downstream primer: 5'-GTTCGTAGATACAAAGTAAG-3' (as shown in SEQ ID NO. 12).

[0062] 7. Bioinformatics analysis of chicken miRNA SNPs:

[0063] The SNPs on mature chicken miRNAs were analyzed using online software, miRNA-SNiPer ( http: / / www.integratomics-time.com / miRNA-SNiPer / ) and dbSNP( http: / / www.ncbi.nlm.nih.gov / snp ) database, identified SNP ID rs14098602, miR-3528 SNP site in the precursor region, chromosome 17 (Chr17). http: / / www.mirbase.org / database (20.0), the miR-3528 SNP site has a host gene, and the name of the host gene is ENSGALT00000042574; SNORA17-201; exon 1.

[0064] 8. Verification of SNPs in Gushi-Anka Chicken Populations:

[0065] Using the mixed DNA pool method, 100 individuals of Gushi chicken-Anka chicken F2 were mixed into one pool. Four pairs of synthetic primers were used for amplification and the products were directly sequenced. The sequencing results were analyzed for SNPs using DNA STAR, BioXM (2.0) and Primer 5.0. The sequencing results showed that the mutation sites were located where there were overlapping peaks. The DNA pool was used to verify the existence of the chicken miRNA SNP. The +12bp A>G mutation in the precursor region of miRNA-3528 was detected. The position on the sequencing map was 88bp (see Figure 1 ).

[0066] Specifically, the nucleotide sequence of the miRNA-3528 gene SNP molecular marker is shown in SEQ ID NO. 1, and the 12th base from the 5' end is A or G. The nucleotide sequence shown in SEQ ID NO. 1 is located at positions 8329802 to 8329898 of the chicken reference genome (GRCg7b version), and the miRNA-3528 gene SNP molecular marker is located at position 8329813 of the chicken reference genome (GRCg7b version).

[0067] 2. Association analysis between miRNA-3528 gene SNP molecular markers and economic traits

[0068] 1. Primer design and synthesis:

[0069] The primers designed and synthesized for the mutation site in the complementary region of the mature miRNA-3528 gene (rs14098602A / G) are as follows:

[0070] Upstream primer F PRimer: 5'-ACGTTGGATGCATGGCACTACAGCCATATC-3' (as shown in SEQ ID NO. 2);

[0071] Downstream primer R PRimer: 5'-ACGTTGGATGTGAGTCGCAGTGGATACAAG-3' (as shown in SEQ ID NO. 3);

[0072] Single base extension primer: 5'-GCTCATGCATTACACAG-3' (as shown in SEQ ID NO. 4);

[0073] Annealing temperature Tm: 45.6°C.

[0074] 2. Genotype analysis:

[0075] SequenomMassARRay time-of-flight mass spectrometry was used to genotype different individuals in the F2 generation resource group, and MassARRayTypeR software was used to analyze the mass spectra (this process was completed by Beijing Hyster Clinical Research Institute Co., Ltd.).

[0076] The purified genomic DNA sample was quantitatively diluted to 100 ng / μL. 1 μL of sample was added to each well of a 384-well plate according to the designed primer sequence. Amplification was then performed according to the following reaction system and procedure. After the reaction was completed, 0.3 U of shrimp alkaline phosphatase (SAP) was added to remove the remaining dNTPs. Single-base extension reactions were then performed. The reaction products were desalted with resin and spotted on a chip using an automatic spotter. The spotted chip was then detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

[0077] a. PCR reaction system and reaction program settings

[0078] The PCR reaction system is shown in Table 1, and the PCR amplification reaction procedure is as follows:

[0079] Pre-denaturation: 95°C for 2 min; 95°C for 30 s; 56°C for 30 s; 72°C for 60 s, 45 cycles; 72°C for 5 min; and insulation at 25°C.

[0080] Table 1 PCR total reaction system

[0081]

[0082]

[0083] b. SAP enzyme digestion reaction

[0084] The SAP enzyme digestion reaction system is shown in Table 2. SAP enzyme digestion was performed in a PCR instrument. The digestion reaction program was: 37°C for 40 min; 85°C for 5 min; and insulation at 25°C.

[0085] Table 2 SAP enzyme digestion reaction

[0086]

[0087] c. Single base extension reaction

[0088] The single base extension reaction system is shown in Table 3. The single base extension reaction was performed in a PCR instrument with the following reaction program: initial denaturation at 94°C for 30 s; 40 cycles of 94°C for 5 s, 52°C for 5 s, and 80°C for 5 s × 5 inner cycles, and 72°C for 3 min; and insulation at 25°C.

[0089] Table 3 Single base extension reaction system

[0090]

[0091] d. Resin desalination

[0092] The reaction products were desalted with resin for 20 minutes. The desalting procedure involved adding 16 μL of water to the corresponding wells of the extension reaction product, adding 6 mg of clean rosin resin to the reaction product, sealing the membrane, and rotating vertically at low speed for 20 minutes to allow for full contact between the resin and the reaction product. Centrifugation at 3200 rpm for 5 minutes allowed the resin to settle to the bottom of the wells. The desalted samples were transferred from the 384-well plate to a Sequenom chip using an automated spotter for spotting.

[0093] e. Flight mass spectrometry detection of reaction products

[0094] The data were directly analyzed by flight mass spectrometry instrument to perform genotyping of the miRNA-3528 gene (rs14098602A / G) SNP site, and three genotypes, AA, AG, and GG, were obtained. The characteristic mass spectra of different genotypes are shown in Figure 2 .

[0095] Specifically, the basic principle of SNP typing by the MassARRAY system is to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology in combination with a single primer extension reaction, which is achieved by directly identifying the molecular weight of nucleic acid fragments. After synthesis and dilution, the primer sequence is first subjected to multiple PCR amplification to increase the template for the single base extension reaction, and then the single base extension reaction is carried out. ddNTP is used in the single base extension reaction, so the reaction automatically terminates after one base is extended. Finally, the extension product is added to the chip using a fully automatic spotting machine and mass spectrometry typing is performed by MassARRAY. The MASSARRAY platform has the following advantages in detecting SNPs: accurate and reliable, directly detecting the molecular weight, without involving fluorescent labels, gel electrophoresis, etc., it can detect the difference of one base, with high accuracy, and the probability of error of the machine itself is very low. There is no need for re-verification and no need to design statistical repetitions. The throughput is high and can be applied to the detection of several to hundreds of SNP sites. The detection time is short. Using mass spectrometry detection, 380 samples can be tested within 40 minutes, and the test results are displayed in real time.

[0096] 3. Statistical analysis of the polymorphism frequency of SNP sites in the chicken miRNA-3528 gene:

[0097] Analysis of population genetic polymorphism: The genetic structure of the population was analyzed using Excel based on the population genotype data, and the polymorphic information content (PIC), population heterozygosity (He) and effective number of alleles (Ne) of the gene were estimated.

[0098] Allele frequency refers to the relative ratio of a certain gene to its allele in a population, with a value ranging from 0 to 1, that is:

[0099] Allele frequency = number of a certain gene / total number of genes at the same locus in the population.

[0100] Genotype frequency refers to the ratio of the number of individuals with a certain genotype in a population to the total number of the population, that is:

[0101] Genotype frequency = total number of individuals with a certain genotype / total number of the measured population.

[0102] The statistical analysis results of the genotype frequency of the A>G locus of miRNA-3528 in the F2 resource population are shown in Table 4, and the accumulation of individuals with different genotype typings of miR-3528 is as follows Figure 3 . The results show that in the F2 resource population, 437 are of the AA genotype, 214 are of the AG genotype, and 23 are of the GG genotype. The genotype frequencies of AA, AG, and GG are 0.6484, 0.3175, and 0.034 respectively. The frequencies of the A and G alleles are 0.8071 and 0.1929 respectively. The gene frequency of allele A of miR-3528 (+12bp A>G) exceeds 0.8, which is the dominant gene.

[0103] The mutation site of chicken miRNA-3528 (+12bp A>G) is moderately polymorphic with 0.25 < PIC < 0.5, and there are three genotypes. The statistical results of the population genetic polymorphism information parameters of the mutation site are polymorphism information content PIC (0.2629), heterozygosity He (0.3114), and effective allele number Ne (1.4521). Therefore, a correlation analysis was conducted with the relevant economic traits of the resource population.

[0104] Table 4 Genotype frequencies and gene frequencies of miRNA-3528 gene in the F2 generation resource population

[0105]

[0106]

[0107] 4. Influence of miRNA-3528 SNP locus on miRNA secondary structure:

[0108] Using the Mfold online software, the secondary structure of the miR-3528 (+12bp A>G) allele was predicted, and the change in energy value caused by SNP was not significant. The specific results are shown in Table 5 and Figure 4The SNP at miR-3528 (+12bp A>G) had no significant effect on the secondary structure of its own miRNA, and the SNP at miR-3528 (+12bp A>G) did not change the minimum free energy value of its secondary structure, suggesting that this SNP site did not affect the processing of its miRNAs.

[0109] Table 5 Effects of chicken miRNA SNP on secondary structure energy value

[0110]

[0111] 5. Association analysis between the polymorphism of the chicken miRNA-3528 gene SNP site and chicken body weight traits:

[0112] Association analysis model: SPSS (20.0) software was used to analyze the correlation between gene SNP sites and economic traits of chickens. The data for each trait were ensured to be normally distributed, and the least squares method was then used to correct the data. Based on the data characteristics, a general linear model was used to analyze the genotype effect and compare the differences between the genotypes. The results are shown in Tables 7 to 11. Model I is the association analysis model for the polymorphism of this site and economic traits, namely:

[0113] Model I:Y ijkl =μ+G i +S j +H k +f l +e ijklm

[0114] Model II:

[0115] Among them, Y ijkl is the individual phenotypic value, μ is the population mean, G i is the marker genotype effect (i=3), f l is the family effect (1=7), s j is the gender effect (j=2), H k is the batch effect (k=2), e ijklm is the random error, G i 、s j 、H k is a fixed factor, f l The Borferroin method was used to control multiple comparisons, and P < 0.05 was considered significant.

[0116] ① Association analysis with carcass traits of the F2 generation resource group was performed. The results are shown in Table 6. The results showed that the SNP polymorphism of the chicken miRNA-3528 gene was significantly associated with chicken carcass traits (12 weeks of age). The eviscerated weight of individuals with the GG genotype was significantly greater than that of individuals with the AA and AG genotypes (GG>AG>AA, p<0.05).

[0117] Table 6 Association analysis between different genotypes of miR-3528 (+12bp A>G) mutation site and carcass traits

[0118]

[0119]

[0120] Note: * represents p<0.05; the same letters indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05).

[0121] ② Association analysis with growth traits of the F2 generation resource group was conducted. The results are shown in Table 7. The results showed that the chicken miRNA-3528 gene SNP single nucleotide polymorphism was significantly associated with chicken weight. The GG genotype significantly increased birth weight (GG>AG>AA, p<0.05), weight at 2 weeks of age (GG>AG>AA, p<0.05), and weight at 4 weeks of age (GG>AG, GG>AA, p<0.05). Using this genotype as an auxiliary selection and molecular genetic breeding marker for birth weight, weight at 2 weeks of age, and weight at 4 weeks of age can rapidly establish a flock with superior genetic resources. Furthermore, weight at 6, 8, 10, and 12 weeks of age was higher in the GG genotype than in the AA and AG genotypes.

[0122] Table 7 Association analysis between different genotypes of miR-3528 (+12bp A>G) mutation site and growth traits

[0123]

[0124] Note: ** represents p<0.01, * represents p<0.05; the same letters indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05).

[0125] ③ Association analysis was performed with the size traits of the F2 generation resource population, and the results are shown in Table 8. The results showed that the SNP single nucleotide polymorphism of the chicken miRNA-3528 gene was associated with the body size traits of chickens. The birth shank length, shank length at 4 weeks, shank length at 8 weeks, shank length at 12 weeks, shank circumference at 4 weeks, shank circumference at 8 weeks, shank circumference at 12 weeks, chest depth at 8 weeks, chest depth at 12 weeks, chest width at 8 weeks, sternum length at 4 weeks, body oblique length at 8 weeks, body oblique length at 12 weeks, pelvic width at 4 weeks, pelvic width at 8 weeks, and pelvic width at 12 weeks were all greater for the GG genotype than for the GA genotype and the AA genotype.

[0126] Table 8 Association analysis between different genotypes of miR-3528 (+12bp A>G) mutation site and body size traits

[0127]

[0128]

[0129] Note: 1) The same letters indicate no significant difference (p>0.05), and different letters indicate significant difference (p<0.05).

[0130] 2) 0, 4, 8, and 12 represent weeks. SL = shin length; SG = shin circumference; CD = chest depth; CB = chest breadth; BBL = sternal length; PA = pectoral angle; BSL = body oblique length; PB = pelvic breadth.

[0131] ④ Association analysis was performed with the F2 generation resource population size traits. The results are shown in Table 9. The results showed that the chicken miRNA-3528 gene SNP single nucleotide polymorphism was significantly associated with chicken quality traits. The GG genotype significantly reduced the skin fat rate (GG<AG<AA, P<0.05); the GG genotype significantly increased the breast muscle density (GG>AG>AA, p<0.05); the GG genotype significantly increased the breast muscle area ratio (GG>AG>AA, p<0.05); the leg muscle area ratio, The GG genotype has an increased incidence rate (GG>AG>AA); the width of intermuscular fat and the thickness of subcutaneous fat are both AG<GG<AA, the pH of chest muscle and leg muscle are the smallest in the GG genotype (GG<AG<AA); the body weight after leg hair removal is the largest in the GG genotype (GG>AG>AA); the leg muscle density (GG>AA) and pancreatic weight rate (GG>AA, GG>AG) are all higher in the GG genotype than in the AA genotype; the chest muscle fiber diameter is smaller in the GG genotype than in the AA genotype (GG<AG).

[0132] Table 9 Association analysis between different genotypes of miR-3528 (+12bp A>G) mutation site and meat quality traits

[0133]

[0134] Note: * represents p<0.05; a, b represent significant differences among different genotypes within the same indicator (p<0.05).

[0135] Example 2 Application of a Chicken miRNA-3528 Gene SNP Molecular Marker in Evaluating Chicken Blood Biochemical Indices

[0136] Blood biochemical indicators are crucial to the normal growth and development of animals. They can directly reflect the physiological functions of animals and play an important indicative role. In this example, the polymorphism of the chicken miRNA-3528 gene SNP molecular marker was associated with the F2 generation resource group for chicken serum enzymes. The results are shown in Table 10. The results show that the chicken miRNA-3528 gene SNP single nucleotide polymorphism was significantly associated with chicken serum enzymes. The GG genotype of albumin ALB, globulin GLO, and cholinesterase CHE were significantly lower than the AG and AA genotypes (GG < AG < AA, p < 0.05); the GG genotype of total protein TP was significantly lower than the AG and AA genotypes (GG < AG, GG < AA, p < 0.05); the GG genotype of lactate dehydrogenase LDH was significantly lower than the AG genotype and AA genotype (GG < AG, GG < AA, p < 0.05); the GG genotype of alanine aminotransferase ALT, aspartate aminotransferase AST, creatinine CREA, total cholesterol CHO, triglycerides TG, high-density lipoprotein HDL, and creatine phosphokinase CK were all higher than the AG genotype and AA genotype (GG> AG> AA).

[0137] Table 10 Association analysis between different genotypes of miR-3528 (+12bp A>G) mutation site and serum enzymes

[0138]

[0139] Note: ** represents p<0.01, * represents p<0.05; a, b represent significant differences among different genotypes within the same indicator (p<0.05).

[0140] Among them, the content of total protein (TP) is related to protein utilization and feed conversion rate. Low total protein levels indicate that the body is more efficient in metabolizing protein, reducing nitrogen excretion and metabolic waste. Low protein decomposition requirements can use more nutrients for muscle growth, thereby improving feed conversion rate. The content of albumin (ALB) is related to muscle growth and energy metabolism. Low albumin reflects rapid protein turnover, preferentially supporting muscle tissue synthesis and accelerating weight gain. In addition, low albumin reduces the energy consumption of maintaining osmotic pressure and allocates energy to growth-related metabolism. Globulin (GLO) is related to immune stress and resource allocation. Reduced globulin reduces overactivation of the immune system and reduces the energy consumption of immune metabolism. In addition, low globulin content can save energy and nutrients, which can be prioritized for growth and muscle development, thereby improving production performance. Cholinesterase (CHE) is related to immune stress and neuromuscular coordination. Low cholinesterase activity can prolong the action time of acetylcholine, improve feed intake efficiency and exercise capacity. Lactate dehydrogenase (LDH) is related to meat quality. Low LDH levels can reduce lactic acid accumulation, enhance exercise endurance, and support the continuous activity and growth of chickens. Reducing lactic acid accumulation can also reduce muscle acidity and improve meat quality after slaughter.

[0141] Furthermore, mild elevations within the normal range of some blood biochemical indices may reflect the body's increased metabolic demands, tissue repair, or adaptive responses. Elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) within the normal physiological range reflect that the liver is efficiently processing toxins, drugs, or metabolites (such as protein synthesis and gluconeogenesis). γ-Glutamyl transpeptidase (GGT) participates in antioxidant defense in glutathione metabolism; mild elevations may enhance cellular resistance to oxidative stress, help scavenge free radicals, and improve detoxification function. Elevated alkaline phosphatase (AKP) can promote bone growth or repair, bone mineralization, and osteoblast activity; it participates in lipid absorption and intestinal barrier protection. Creatinine is a product of muscle metabolism. In people with well-developed muscles, creatinine levels are close to the upper limit of normal, reflecting high muscle mass and active metabolism.

[0142] Furthermore, among the lipid-related indicators in blood biochemical parameters, high-density lipoprotein (HDL) has a protective effect on cardiovascular health. A slight increase within the normal range can help reverse cholesterol transport and reduce the risk of atherosclerosis. A slight increase in total cholesterol or low-density lipoprotein (LDL) helps support cell membrane repair, the synthesis of steroid hormones (such as sex hormones and cortisol), and provides a reserve when energy demand increases. A slight increase in creatine phosphokinase (CK) indicates enhanced muscle repair and adaptability, promoting muscle growth. CK participates in energy storage (phosphocreatine system), and an increase may reflect increased muscle energy demand and active energy metabolism. Glucose and amylase levels, on the other hand, remain essentially unchanged, indicating good physiological regulation.

[0143] In summary, the GG genotype of the miRNA-3528 gene SNP molecular marker significantly optimizes protein, lipid and energy metabolism by regulating the activity of key enzymes, directly improving the growth rate, feed efficiency and health level of chickens, and promoting the development of an efficient and sustainable intensive breeding industry.

[0144] In summary, the present invention first screened and verified miRNA SNP sites. Using chicken whole genome DNA as a template, primers were designed to amplify the target fragment including the miRNA-3528 precursor. After purification and sequencing, it was found that there was an A or G base polymorphism at the 12th position of the chicken miRNA-3528 gene. Then, specific primers were designed to amplify the gene fragment containing the polymorphic site, and the MassARRAY system matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology combined with a single primer extension reaction was used to perform miRNA SNP analysis on a large number of samples from the F2 generation resource group of Gushi chicken and Anka chicken. Genotyping of the single SNP locus A-3528 (rs14098602A / G) and association analysis with weight traits in the F2 generation resource population revealed a significant association between the miRNA-3528 gene SNP and chicken body weight. The GG genotype significantly increased eviscerated weight, birth weight, two-week-old weight, and four-week-old weight (p<0.05). Using this genotype as a marker for chicken body weight in supplementary selection and molecular breeding can rapidly establish flocks with superior genetic resources. This detection method is accurate, reliable, rapid, sensitive, and cost-effective, making it suitable for supplementary selection and molecular breeding of chickens, playing a crucial role in improving economic traits.

[0145] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of a chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding, characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 12th base from the 5' end is A or G; the economic traits are carcass traits and / or growth traits and / or meat quality traits; the carcass traits include eviscerated weight; the growth traits include birth weight, 2-week weight, 4-week weight, and 6-week weight; the meat quality traits include sebum percentage and breast muscle density.

2. The use of the chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding according to claim 1, characterized in that: The genotype of the SNP molecular marker was detected, and individuals with the SNP molecular marker GG genotype were selected.

3. The use of the chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding according to claim 2, characterized in that: The detection comprises the following steps: using the chicken DNA to be detected as a template, performing PCR amplification, adding a single base extension primer to perform a single base extension reaction, and performing mass spectrometry detection on the reaction product.

4. The use of the chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding according to claim 3, characterized in that: The nucleotide sequences of the PCR primers are shown in SEQ ID NOs. 2-3.

5. The use of the chicken miRNA-3528 gene SNP molecular marker in chicken economic trait improvement breeding according to claim 4, characterized in that: The nucleotide sequence of the single-base extension primer is shown in SEQ ID NO.

4.

6. Use of the chicken miRNA-3528 gene SNP molecular marker according to any one of claims 3 to 5 in improving and breeding chicken economic traits, characterized in that: The detection also includes a SAP enzyme digestion reaction after PCR amplification.

7. An application of a chicken miRNA-3528 gene SNP molecular marker in evaluating chicken blood biochemical indicators for non-disease diagnosis purposes, characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the 12th base from the 5' end is A or G; the blood biochemical indicators include total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase.

8. The use of the chicken miRNA-3528 gene SNP molecular marker according to claim 7 in evaluating chicken blood biochemical indicators, characterized in that: The genotype of the SNP molecular marker was detected. When the genotype of the chicken to be tested was GG, the chicken to be tested was an individual with low total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase; when the genotype of the chicken to be tested was AA, the chicken to be tested was an individual with high total protein, albumin, globulin, cholinesterase, and lactate dehydrogenase.

9. The use of the chicken miRNA-3528 gene SNP molecular marker according to claim 8 in evaluating chicken blood biochemical indicators, characterized in that: Individuals with the SNP molecular marker GG genotype were selected.

Images