Chicken g3bp1 gene snp molecular marker, application thereof, detection primer and detection kit

Abstract

The present application relates to a chicken G3BP1 gene SNP molecular marker and its application, a detection primer and a detection kit. The SNP molecular marker is composed of two molecular markers; the nucleotide sequence of the SNP molecular marker 1 is shown as SEQ ID NO. 1, and the 501th base at the 5' end is C or A; the nucleotide sequence of the SNP molecular marker 2 is shown as SEQ ID NO. 2, and the 501th base at the 5' end is G or A. The present application first discovers two SNP molecular markers on the chicken G3BP1 gene, the site of SNP1 is rs314886487, and there is a C>A mutation, and the site of SNP2 is rs315630742, and there is a G>A mutation. The above two SNP molecular markers have not been reported, are new chicken SNP molecular markers, and are significantly related to chicken fat traits, and can be used as molecular markers for genetic improvement of chicken breeds.

Description

Technical Field

[0001] This invention relates to a chicken G3BP1 SNP molecular markers and their applications, detection primers, and detection kits belong to the field of bio-breeding technology. Background Technology

[0002] Chickens are an important economic poultry species and also a crucial model organism for scientific research. my country is a major producer and consumer of chicken meat, ranking second globally in consumption. Annual chicken sales are second only to pork, highlighting the vital role chickens play in my country's meat production and consumption. Consequently, the intensive and large-scale farming methods for broilers in my country have led to unprecedented growth rates. With increasing chicken sales, consumer demands for quality have also risen. Juicy, tender, and flavorful chicken is particularly popular. However, the increased growth rate of broilers has resulted in a decline in chicken quality. Therefore, improving the quality of chicken products to meet consumer demands has become one of the most pressing issues for my country's broiler industry.

[0003] Numerous studies have shown that the differential distribution of fat in broilers can have drastically different effects on chicken quality. Increasing the intramuscular fat (IMF) content in meat products can effectively increase the juiciness, tenderness, and flavor of the meat, thereby improving meat quality. On the other hand, abdominal fat (AF) is considered a production waste, which reduces feed utilization efficiency and restricts production efficiency. High intramuscular fat and low abdominal fat are important goals in the field of broiler breeding worldwide.

[0004] Intramuscular fat deposition in livestock and poultry is regulated by factors such as genetics, environment, and feed nutrition levels, with genetic factors playing a crucial role. The heritability of intramuscular fat in chickens is 0.1–0.16, and related studies have shown that genetic selection can effectively increase the content of intramuscular fat in chickens. Therefore, developing molecular genetic markers for chicken intramuscular fat and conducting marker-assisted selection breeding can effectively enhance selection intensity, improve the accuracy of genetic assessment of intramuscular fat traits, and accelerate the improvement of intramuscular fat traits.

[0005] Mark-assisted selection (MAS) is a novel strategy for artificial breeding that relies on molecular genetic markers to assist in selecting individuals with specific traits. By fully utilizing information from phenotypes, pedigrees, and genetic markers, it enables early selection, shortens generation time, increases selection intensity, and significantly improves genetic progress. Marker-assisted selection technology is mainly divided into two types: Type I markers and Type II markers. Type I markers are primarily single genes, mainly used to compare the relative distances and linkage and linear relationships of homologous loci among different varieties. Type II markers are mainly DNA loci and fragments with high polymorphism and rich information content. The types of molecular markers include, but are not limited to, single nucleotide polymorphisms (SNPs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPO), amplified fragment length polymorphisms (AFLPs), and microsatellites.

[0006] Single nucleotide polymorphisms (SNPs) are a common type of DNA molecular marker, referring to the insertion, deletion, translocation, amplification, and substitution of a single base in the genome. Studies have found that polymorphisms occurring in introns, exons, 3'UTR, and 5'UTR regions of genes can potentially affect gene function, leading to changes in animal phenotypic traits. Correlation analysis between SNP polymorphisms and meat quality traits can provide auxiliary marker selection for molecular selection breeding in poultry.

[0007] Chinese invention patent CN116656838A, published on August 29, 2023, discloses a SNP molecular marker related to intramuscular fat and body weight in chickens and its uses. Specifically, it discloses three SNP molecular markers on the exons of the LIPE gene, and through correlation analysis between related variant sites and intramuscular fat content and body weight in chickens, it was found that the three SNP molecular markers on the exons of the LIPE gene are significantly correlated with intramuscular fat content and body weight in chickens.

[0008] Although there are SNP molecular markers related to intramuscular fat in chickens in the existing technology, the number is relatively small. Therefore, it is necessary to develop candidate genes related to chicken fat traits, find new molecular markers, and accelerate the genetic improvement process with the help of molecular biology methods, thereby shortening the breeding time and speeding up the breeding process. Summary of the Invention

[0009] The first objective of this invention is to provide a chicken G3BP1 gene This provides a novel SNP molecular marker associated with chicken fat traits, based on existing technologies.

[0010] The second objective of this invention is to provide the application of the aforementioned SNP molecular markers in the breeding of chicken fat traits, thereby providing a new molecular biology approach to accelerate the process of chicken genetic improvement.

[0011] The third objective of this invention is to provide a detection primer for detecting the above-mentioned SNP molecular marker genotype, thereby providing a primer with good specificity for detecting the above-mentioned SNP molecular marker in the prior art.

[0012] The fourth objective of this invention is to provide a kit for detecting the above-mentioned SNP molecular marker genotypes, thereby providing a simple and specific detection method for the rapid detection of the above-mentioned SNP molecular markers.

[0013] To achieve the above objectives, a chicken in this invention... G3BP1 gene The technical solution for SNP molecular markers is: A type of chicken G3BP1 gene The SNP molecular markers consist of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 501st base from the 5' end being C or A; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 501st base from the 5' end being G or A.

[0014] The beneficial effects of the above technical solution are as follows: This invention provides a chicken... G3BP1 gene SNP molecular markers are a pioneering invention. This invention is the first to discover SNPs in chickens. G3BP1 Two SNP markers on intron 11 of the gene were discovered: SNP marker 1, located at rs314886487, contains a C>A mutation, and SNP marker 2, located at rs315630742, contains a G>A mutation. These two SNP markers are newly discovered chicken SNP markers and have not been previously reported.

[0015] To achieve the above objectives, the chicken in this invention G3BP1 The technical solution for applying gene SNP molecular markers in chicken fat trait improvement breeding is as follows: Application of an SNP molecular marker in breeding for improved fat traits in chickens.

[0016] The beneficial effects of the above technical solution are as follows: Through analysis and verification, this invention newly discovers a method for... (in chickens) G3BP1 gene The SNP molecular markers on chickens are significantly correlated with the fat trait and can be used as molecular markers for the genetic improvement of chicken breeds. They can be used for chicken-assisted selection and molecular breeding, which is beneficial for the genetic improvement of chicken populations with superior traits and is of great significance for improving the quality of chicken meat.

[0017] As a further improvement, for the fatty trait being intramuscular fat, the genotype of the SNP molecular marker is detected, and individuals with SNP molecular marker 1 being CC or CA genotype are selected, or individuals with SNP molecular marker 2 being GG or GA genotype are selected.

[0018] Specifically, in practical applications, the type of the nucleotide at position 501 from the 5' end of the sequence shown in SEQ ID NO.1 can be directly detected, and chicken individuals with a CC homozygous or CA heterozygous nucleotide at this position can be selected to establish a chicken population with excellent genetic resources. Alternatively, the type of the nucleotide at position 501 from the 5' end of the sequence shown in SEQ ID NO.2 can be detected, and chicken individuals with a GG homozygous or GA heterozygous nucleotide at this position can be selected to establish a chicken population with excellent genetic resources.

[0019] As a further improvement, the fatty trait is intramuscular fat, sebum, and sebum percentage. The genotype of the SNP molecular marker is detected, and individuals with SNP molecular marker 2 being GG or GA genotype are selected.

[0020] Specifically, in practical applications, the type of nucleotide at position 501 from the 5' end of the sequence shown in SEQ ID NO.2 can be directly detected, and chicken individuals with this site being GG homozygous or GA heterozygous can be selected to establish a chicken population with excellent genetic resources.

[0021] To achieve the above objectives, the technical solution for the detection primers used in this invention for detecting SNP molecular marker genotypes is as follows: A detection primer for detecting SNP molecular marker genotypes, wherein the molecular marker is SNP molecular marker 1, and the nucleotide sequence of the detection primer is shown in SEQ ID NO. 3-4; and wherein the molecular marker is SNP molecular marker 2, and the nucleotide sequence of the detection primer is shown in SEQ ID NO. 5-6.

[0022] The beneficial effect of the above technical solution is that the above primers can effectively amplify the contents of chicken. G3BP1 Fragments of gene SNP molecular markers, assisted gene sequencing, and other methods can be used to quickly detect and screen chicken individuals for genetic improvement.

[0023] To achieve the above objectives, the technical solution of the kit for detecting SNP molecular marker genotypes in this invention is as follows: A kit for detecting SNP molecular marker genotypes, the kit comprising detection primers with nucleotide sequences as shown in SEQ ID NO. 3-4 and / or detection primers with nucleotide sequences as shown in SEQ ID NO. 5-6.

[0024] The beneficial effect of the above technical solution is that the primers for detecting the SNP molecular markers are prepared into a kit, which makes it convenient and quick for users to detect the genotype of SNP molecular markers.

[0025] As a further improvement, the kit also includes one or more of dNTPs, PCR reaction buffer, and DNA polymerase.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to discover in chickens G3BP1 Two SNP molecular markers on the gene were discovered: SNP marker 1, located at rs314886487, contains a C>A mutation, and SNP marker 2, located at rs315630742, contains a G>A mutation. These two SNP molecular markers are newly discovered chicken SNPs and have not been previously reported. Further analysis showed that these two SNP sites are significantly associated with fatty traits in chickens (mainly intramuscular fat) and can serve as molecular markers for genetic improvement of chicken breeds.

[0027] 2. This invention is aimed at chickens G3BP1 Specific PCR primers are designed to amplify SNP molecular markers of genes. The amplified fragments are then sequenced to obtain the genotype of the locus, enabling accurate, rapid, time-saving, and labor-saving detection of the SNP molecular marker. Attached Figure Description

[0028] Figure 1 In Experimental Example 2 of this invention G3BP1 Agarose gel electrophoresis image of gene fragment 1 PCR amplification; Figure 2 In Experimental Example 2 of this invention G3BP1 Gene fragment 2PCR amplification agarose gel electrophoresis image; Figure 3 In Experimental Example 2 of this invention G3BP1 Heterozygous mutation map of gene 314886487C>A site; Figure 4 In Experimental Example 2 of this invention G3BP1 Heterozygous mutation map of the G>A site in gene 315630742. Detailed Implementation

[0029] Studies have shown that increasing the intramuscular fat (IMF) content in meat products can effectively increase the juiciness, tenderness, and flavor of the meat, thereby improving meat quality. However, existing technologies have limited molecular markers related to chicken intramuscular fat. Therefore, this invention provides a chicken... G3BP1The gene SNP molecular marker consists of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 501st base from the 5' end being C or A; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 501st base from the 5' end being G or A.

[0030] This invention was first discovered in chickens G3BP1 Two SNP molecular markers on the gene were discovered: SNP marker 1, located at rs314886487, contains a C>A mutation, and SNP marker 2, located at rs315630742, contains a G>A mutation. These two SNP molecular markers are newly discovered chicken SNPs and have not been previously reported. Further analysis showed that these two SNP sites are significantly associated with the fat trait in chickens (mainly intramuscular fat) and can serve as molecular markers for genetic improvement of chicken breeds.

[0031] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; however, these embodiments are merely examples and do not constitute any limitation on the scope of the present invention. Modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent manufacturers.

[0032] Laboratory animals: This study used a complete phenotypic record of 766 Gushi-Anka F2 chicken breeds as the research subject. This breed was constructed by the Henan Provincial Engineering Technology Research Center for Poultry Germplasm Resources Innovation using the slow-growing local breed Gushi chicken and the fast-growing introduced meat breed Anka chicken. It included four orthogonal cross families (Anka roosters mated with Gushi hens) and three reciprocal cross families (Gushi roosters mated with Anka hens). The chickens were raised under the same feeding and management conditions at the Henan Agricultural University Experimental Chicken Farm, and the slaughter experiment was conducted at the Henan Agricultural University Poultry Genetic Improvement Laboratory.

[0033] Nutritional levels during the rearing period: 0-4 weeks old: energy level 12.40 MJ / kg, protein level 20.1%; 5-8 weeks old: energy level 12.70 MJ / kg, protein level 18.2%; 9-12 weeks old: energy level 12.75 MJ / kg, protein level 16.0%. Chickens of different family lines were mixed, caged, with free access to feed and water.

[0034] A type of chicken G3BP1 Example 1 of gene SNP molecular markers The chicken in this embodiment G3BP1 The gene SNP molecular marker consists of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 501st base from the 5' end being C or A; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 501st base from the 5' end being G or A.

[0035] SNP molecular marker 1 corresponds to the sequence information of the chicken reference genome GRCg6a version published by NCBI, at deoxynucleotide position 314886487 on chromosome 13 (i.e., rs314886487).

[0036] SNP molecular marker 2 corresponds to the sequence information of the chicken reference genome GRCg6a version published by NCBI, at deoxynucleotide position 315630742 on chromosome 13 (i.e., rs315630742).

[0037] chicken G3BP1 Example 1: Application of gene SNP molecular markers in breeding for improved fat traits in chickens This embodiment describes the application of SNP molecular markers in the breeding of chickens to improve the fat trait. The fat trait refers to intramuscular fat. Genotypes of the SNP molecular markers are detected, and individuals with SNP molecular marker 1 having the CC or CA genotype, or individuals with SNP molecular marker 2 having the GG or GA genotype, are selected to establish a chicken population with superior genetic resources. The fat trait also refers to intramuscular fat, subcutaneous fat, and subcutaneous fat percentage. Genotypes of the SNP molecular markers are detected, and individuals with SNP molecular marker 2 having the GG or GA genotype are selected to establish a chicken population with superior genetic resources.

[0038] Example 1 of a detection primer for detecting SNP molecular marker genotypes The nucleotide sequences of the detection primers used in this embodiment to detect the SNP molecular marker 1 genotype are shown below: F Primer:5'-TGGCTTCTGGGACATTCCCTCCA-3' (shown in SEQ ID NO.3); R Primer:5'-GTGATAGAGAGGGTCTGCTGTT-3' (shown in SEQ ID NO.4).

[0039] The nucleotide sequences of the detection primers used to detect the SNP molecular marker 2 genotype are shown below: F Primer:5'-CTCCAAATCCTGGCTTCT-3' (shown in SEQ ID NO.5); R Primer:5'-GAGGGTCTGCTGTTCTTCAT-3' (shown in SEQ ID NO.6).

[0040] Example 1 of a kit for detecting SNP molecular marker genotypes The kit for detecting SNP molecular marker genotypes in this embodiment includes detection primers with nucleotide sequences as shown in SEQ ID NO. 3~6, ​​dNTPs, PCR reaction buffer, and DNA polymerase.

[0041] Example 1: Extraction and Detection of Chicken Genomic DNA In this experiment, genomic DNA was extracted from the F2 generation of the Gushi-Anka chicken population, and the concentration of the extracted genomic DNA was detected by spectrophotometry. The specific operation steps are as follows: 1. Extraction of genomic DNA Using 766 chickens from the F2 generation of the Gushi-Anka chicken resource population as 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℃. Genomic DNA was extracted from the blood samples of the F2 generation resource population using a blood genomic DNA extraction kit, dissolved in TE buffer, and stored at 4℃ for later use. The specific experimental steps are as follows: (1) Take 20 μL of whole blood into a 1.5 mL centrifuge tube and add 180 μL of GA buffer; (2) Add 20 μL of Proteinase K solution and mix well; (3) Add 200 μL of buffer GB, mix thoroughly by inverting, place at 70°C for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap; (4) Add 200 μL of anhydrous ethanol and shake well for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap. (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube; (6) Add 500 μL of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube. (7) Add 600 μL of washing solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube. (8) Repeat step 7; (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing in the adsorption material. Note: The purpose of this step is to remove residual rinsing solution from the adsorption column. Residual ethanol in the rinsing solution will affect subsequent enzyme reactions (enzyme digestion, PCR, etc.) experiments.

[0042] (10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle of the adsorption membrane, place at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution into the centrifuge tube.

[0043] Note: The elution buffer volume should not be less than 50 μL; a smaller volume will affect the recovery efficiency. The pH value of the elution buffer has a significant impact on the elution efficiency. If ddH2O is used as the elution buffer, its pH value should be maintained within the range of 7.0-8.5; a pH value below 7.0 will reduce the elution efficiency. Furthermore, the DNA product should be stored at -20℃ to prevent DNA degradation. To increase the yield of genomic DNA, the solution obtained after centrifugation can be added back to the CB3 adsorption column, incubated at room temperature for 2 min, and then centrifuged at 12,000 rpm (~13,400 × g) for 2 min.

[0044] 2. Spectrophotometric determination of genomic DNA concentration The absorbance (OD value) of the DNA sample at 260 nm and 280 nm was measured using a UV spectrophotometer. The DNA concentration and the OD260 / OD280 ratio were then calculated. If the OD260 / OD280 ratio was below 1.6, it indicated a possible high level of protein or phenolic compounds in the sample, requiring further purification. If the ratio exceeded 1.8, RNA removal and DNA purification may be necessary. The DNA concentration (ng) was calculated using the formula 50 × OD260 value × dilution factor. After DNA testing, a portion was diluted to 100 ng / μL and stored at -20°C for later use; the remainder was stored at -80°C.

[0045] Experimental Example 2 G3BP1 Detection of gene mutation sites In this experiment, chicken genomic DNA extracted from Experiment 1 was randomly selected, mixed to construct a DNA pool, and then amplified using amplification primers. G3BP1 Gene fragments are amplified and sequenced. The sequencing results are then analyzed. The specific steps are as follows: 1. Construction of the DNA pool 100 DNA samples (concentration of 100 ng / μL) extracted from Experiment 1 were randomly selected, and 0.5 μL of DNA was extracted from each sample and mixed to construct a DNA pool.

[0046] 2. Design of PCR amplification primers (1) PCR amplification G3BP1 Gene fragment 1 Chicken G3BP1 The genomic sequence of the gene in GenBank was used as a reference (GRCg6a (GCF_000002315.6), GeneID:416265). Sequencing PCR primers for the chicken G3BP1 locus rs314886487 were designed using NCBI. The amplified fragment was 561 bp in length. The agarose gel electrophoresis pattern of the PCR product used to detect polymorphism is shown in the image. Figure 1 As shown in the figure, the designed primer pairs have good specificity and are free from primer dimers and stray band interference.

[0047] The nucleotide sequences of the sequencing PCR primer pairs are as follows: F Primer:5'-TGGCTTCTGGGACATTCCCTCCA-3' (shown in SEQ ID NO.3); R Primer:5'-GTGATAGAGAGGGTCTGCTGTT-3' (shown in SEQ ID NO.4).

[0048] (2) PCR amplification G3BP1 Gene fragment 2 Chicken G3BP1 The genome sequence of the gene in GenBank was used as a reference (GRCg6a (GCF_000002315.6), GeneID:416265). Chickens were designed using NCBI. G3BP1 The sequencing PCR primer pair for locus rs315630742 amplified a fragment with a total length of 563 bp. The agarose gel electrophoresis pattern of the PCR product used to detect polymorphism is shown in the image. Figure 2 As shown in the figure, the designed primer pairs have good specificity and are free from primer dimers and stray band interference.

[0049] The nucleotide sequences of the sequencing PCR primer pairs are as follows: F Primer:5'-CTCCAAATCCTGGCTTCT-3' (shown in SEQ ID NO.5); R Primer:5'-GAGGGTCTGCTGTTCTTCAT-3' (shown in SEQ ID NO.6).

[0050] 3. Chicken G3BP1 Cloning of gene fragments Using the DNA pool obtained in step 1 as a template, PCR amplification (Touchdown PCR) was performed using the sequencing PCR primer pairs in step 2. The PCR amplification reaction system was 10 μL. The specific reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0051] Table 1 PCR reaction system Table 2 Touchdown PCR Reaction Procedure 4. Sequencing of PCR products After PCR amplification, electrophoresis was performed on a 2% agarose gel at 120V for 40 min. Ethidium bromide (EB) staining was used for detection, and imaging was performed using a SYNGENE GBOX and Kodak120 gel imaging system (Syngene, USA). The PCR products were then excised, recovered, and purified. The gel containing the target fragment was excised from the agarose gel under UV light and placed in a 2.0 mL centrifuge tube. The PCR products were then purified using a PCR product purification kit (Omega), following the kit's instructions. The purified PCR products were sent to Qingke Biotechnology Co., Ltd. (Beijing) for sequencing.

[0052] The sequencing peak diagram was analyzed and the sequence was aligned. G3BP1 The results of gene fragment 1 are as follows Figure 3 (The arrows in the figure indicate mutation sites.) As shown in the figure, in... G3BP1 The presence of C>A polymorphism in the genes indicates that the chickens screened out... G3BP1 Single nucleotide polymorphisms of genes.

[0053] G3BP1 The results of gene fragment 2 are as follows Figure 4 (The arrows in the figure indicate mutation sites.) As shown in the figure, in... G3BP1 The presence of G>A polymorphism in the genes indicates that the chickens screened out have this condition. G3BP1 Another single nucleotide polymorphism in a gene.

[0054] Experimental Example 3: Chicken G3BP1 Association analysis of gene SNP molecular markers with fatty traits This experiment uses SPSS software to analyze the correlation between SNP molecular markers and fatty traits, and establishes an association analysis model. The specific operation steps are as follows: Based on population genotype statistics, the genotype frequency, gene frequency, Hay-Wen balance (HWpval), and minor allele frequency (MAF) of the target gene were calculated and analyzed. The results are shown in Tables 3 and 4.

[0055] Table 3. Genotype frequency statistics of the C>A locus in the G3BP1 gene. Table 4. Genotype frequency statistics of the G3BP1 gene at the G>A locus (315630742). Association analysis model: The association between different genotypes and related traits in the F2 resource population was analyzed using a mixed linear model based on SPSS 26.0 software. The analysis model is as follows: Y ijklm =µ+G i +S j +H k +f l +e ijklm In the formula: Y ijklm G represents the individual phenotypic value, µ represents the population mean, and G represents the population mean. i To mark the genotype effect (i=3), f l For family effect (l=7), S j For gender effect (j=2), Hk is batch effect (k=2), e ijklm For random error, G i S j H k As a fixed factor, f l The random factor was used; SPSS 26.0 software was used to analyze the correlation between gene loci and fatty traits. Multiple comparisons were performed using the Bonferroni method. p <0.05 indicates a significant difference.

[0056] To ensure that each trait data is normally distributed, the least squares method was used to correct the data. Based on the data characteristics, the genotype effect was analyzed using a multiple linear model and the Benferroni multiple comparison method was used to compare the differences between genotypes. The results are shown in Tables 5 and 6.

[0057] Table 5. Association analysis results between different genotypes of the G3BP1 gene 314886487C>A site and fatty trait. Table 6. Association analysis results between different genotypes of the G3BP1 gene at the 315630742G>A locus and the fatty trait. Note: The same shoulder letter across different lines indicates no significant difference. P >0.05), different letters indicate significant differences ( P <0.05).

[0058] As shown in Table 5, chicken G3BP1 A C>A mutation at the SNP (rs314886487) site was significantly associated with intramuscular fat trait, with CC and CA individuals exhibiting higher intramuscular fat indices than AA individuals, and CA individuals having lower intramuscular fat indices than CC individuals. This indicates that the C allele favors intramuscular fat formation in chickens. G3BP1 The CC and CA genotypes in the single nucleotide polymorphism at the rs314886487 locus of the gene are used as markers of intramuscular fat in chickens and molecular genetic markers to improve chicken meat quality in assisted selection breeding.

[0059] As shown in Table 6, chicken G3BP1 The G>A mutation at the SNP (rs315630742) was significantly associated with adipose tissue traits, specifically: GG and GA individuals had higher fat indices than AA individuals; GA individuals had lower intramuscular fat indices than GG individuals; and GA individuals had higher sebum and sebum percentage indices than GG individuals. This indicates that the G allele favors fat formation in chickens. G3BP1 The GG and GA genotypes in the single nucleotide polymorphism at the rs315630742 site of the gene are used as markers of intramuscular fat in chickens and molecular genetic markers to improve chicken meat quality in assisted selection breeding.

[0060] In summary, this invention has discovered a molecular marker rs314886487 associated with the quantitative trait of intramuscular fat in chickens. Its genotype is significantly correlated with intramuscular fat in chickens. Correlation analysis and significance tests show that the intramuscular fat of chickens with the CC genotype is significantly different from that of chickens with the AA and CA genotypes. Statistically, when the sample genotype is homozygous and the mutant CC, the genotypes with the highest probability of having intramuscular fat content higher than the normal average level are wild-type AA and single mutant CA samples.

[0061] This invention discovered another molecular marker, rs315630742, which is associated with the quantitative trait of intramuscular fat in chickens. Its genotype is highly significantly associated with intramuscular fat in chickens. Correlation analysis and significance tests showed that the intramuscular fat of chickens corresponding to the GG genotype samples was significantly different from that of the AA and GA genotype samples. Statistically, when the sample genotype is homozygous mutant GG, the genotypes with intramuscular fat content higher than the normal average level are wild-type AA and single mutant GA samples.

[0062] This invention designs SNP molecular marker primers and detection methods based on the above-mentioned SNP sites. It only requires simple PCR-specific amplification and Sanger sequencing to identify the high intramuscular fat genotype, which facilitates the detection and screening of chicken strains or breeds with high intramuscular fat and can greatly accelerate the genetic improvement of superior chicken populations.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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. A type of chicken G3BP1 The application of gene SNP molecular markers in breeding for improvement of chicken fat traits is characterized by: The SNP molecular marker consists of SNP molecular marker 1 and SNP molecular marker 2; the nucleotide sequence of SNP molecular marker 1 is shown in SEQ ID NO.1, with the 501st base from the 5' end being C or A; the nucleotide sequence of SNP molecular marker 2 is shown in SEQ ID NO.2, with the 501st base from the 5' end being G or A; the fatty trait is intramuscular fat; the genotype of SNP molecular marker 1 is detected, and individuals with SNP molecular marker 1 having the CC genotype are selected; The genotype of the SNP molecular marker 2 was detected, and individuals with the SNP molecular marker 2 being the GG genotype were selected.

2. The chicken according to claim 1 G3BP1 The application of gene SNP molecular markers in breeding for improved fat traits in chickens is characterized by: The nucleotide sequences of the detection primers for detecting the SNP molecular marker 1 genotype are shown in SEQ ID NO. 3~4.

3. The chicken according to claim 1 G3BP1 The application of gene SNP molecular markers in breeding for improved fat traits in chickens is characterized by: The nucleotide sequences of the detection primers for SNP molecular marker 2 genotype detection are shown in SEQ ID NO.5~6.

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