Application of molecular genetic marker pair in predicting egg weight character

Abstract

The invention discloses application of a molecular genetic marker pair in prediction of egg weight traits. According to the invention, molecular genetic marker pairs of egg weight are excavated, a detection method based on a PCR principle is developed, early accurate prediction of egg weight traits can be realized through double-marker detection, the prediction accuracy is improved, and compared with gene detection methods such as gene chips and re-sequencing, the method has the advantages of simplicity, economy and wide application prospect. The method has important value for reducing breeding cost and accelerating excellent strain breeding.

Description

Technical Field

[0001] This invention relates to the application of molecular genetic marker pairs in predicting egg weight traits, and belongs to the fields of gene detection, poultry breeding and production. Background Technology

[0002] In poultry breeding, egg weight is an important economic trait, and its prediction mainly relies on statistical analysis of phenotypic data and evaluation of pedigree information. Current methods suffer from several technical bottlenecks: first, actual data can only be obtained after hens enter their laying period, leading to long breeding cycles; second, errors can occur during the recording of large amounts of phenotypic data, affecting prediction accuracy; and third, conventional breeding methods have limited capacity to increase egg weight per generation.

[0003] The emergence of marker-assisted selection (MAG) technology has provided a new approach for early prediction. Existing research shows a significant association between specific SNPs (single nucleotide polymorphisms) in the chicken genome and egg production traits, but systematic research on specific molecular markers for egg weight traits is still lacking. Currently available gene detection methods mostly focus on traits such as disease resistance or growth rate, with a scarcity of SNP detection technologies specifically for egg weight prediction, and most rely on single-molecule genetic marker detection. Furthermore, conventional detection techniques such as microarray typing or sequencing have drawbacks such as high equipment requirements and complex operation, hindering their widespread application in poultry farms. Therefore, there is an urgent need for a method that can achieve accurate early prediction of egg weight traits, reducing breeding costs and accelerating the selection of superior strains. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of molecular genetic markers in predicting egg weight traits, so as to predict the egg weight of chickens in an early, accurate, simple and economical way, reduce breeding costs and accelerate the breeding speed.

[0005] Technical Solution: To solve the above-mentioned technical problems, this invention provides the application of molecular genetic marker pairs rs431829386 and rs316872042 in predicting egg weight traits. The molecular genetic marker pairs rs431829386 and rs316872042 are located at positions 7311314 and 7311718 on chromosome 19, respectively (based on the GRCg6a reference genome version). Both rs431829386 and rs316872042 are located at... TRIM37 Gene.

[0006] This invention also provides a detection kit containing the molecular genetic marker pairs rs431829386 and rs316872042 for the prediction of egg weight traits. The molecular genetic marker pairs rs431829386 and rs316872042 are located at positions 7311314 and 7311718 on chromosome 19, respectively (based on the GRCg6a reference genome version). Both the molecular genetic marker pairs rs431829386 and rs316872042 are located at... TRIM37 Gene.

[0007] The molecular genetic markers rs431829386 and rs316872042 are of the AG+GT or AA+GG type.

[0008] Among them, individuals with the genotype AA+GG are relatively high egg weight individuals; individuals with the genotype AG+GT are relatively low egg weight individuals.

[0009] Among them, relatively high egg weight and relatively low egg weight are relative to the average egg weight of all eggs in the group. When the egg weight is greater than the average egg weight, the individual is a relatively high egg weight individual; when the egg weight is less than the average egg weight, the individual is a relatively low egg weight individual.

[0010] The present invention also provides a specific primer pair for the molecular genetic markers rs431829386 and rs316872042, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] The present invention also provides the application of the specific primer pairs of the molecular genetic markers rs431829386 and rs316872042 in the determination of egg weight traits, the nucleotide sequences of the specific primer pairs being shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] The present invention also provides a kit containing specific primer pairs of the molecular genetic markers rs431829386 and rs316872042 for the determination of egg weight traits, the nucleotide sequences of the specific primer pairs being shown in SEQ ID NO.1 and SEQ ID NO.2.

[0013] The application includes the following steps: (1) The hen DNA was amplified by PCR to obtain the amplification product; the nucleotide sequences of the PCR amplification primers are shown in SEQ ID NO.1 and SEQ ID NO.2; (2) Agarose gel electrophoresis was performed, and the amplified product with a band size of 641 bp was sequenced and compared with the nucleotide sequence shown in SEQ ID NO.3; (3) When the genotype at rs431829386 is AA and the genotype at rs316872042 is GG, the tested individual is determined to be a relatively high egg weight individual; when the genotype at rs431829386 is AG and the genotype at rs316872042 is GT, the tested individual is determined to be a relatively low egg weight individual.

[0014] This invention mainly includes the screening of egg weight-related genetic marker pairs, the detection method for these genetic marker pairs, and the verification of the accuracy of the detection method: 1. Screening of this molecular genetic marker pair 1) Phenotypic and genotypic determination: Egg weight at 32 weeks of age was recorded in 3450 Loch Ness Red hens. Genotypic data of 6.45 million SNP loci were obtained for these 3587 individuals using a strategy of SNP genotyping chip combined with reference panel filling.

[0015] 2) Genome-wide association analysis: Using the phenotypic and genotypic data mentioned above as input, and incorporating generational fixed factors, a mixed linear model was used to calculate the statistically significant SNPs associated with the 32-week-old egg weight trait at the genome-wide level. Figure 1 ).

[0016] 3) Identify molecular genetic marker pairs using bioinformatics: Perform fine-grained site annotation on significantly correlated signal regions, indicating that the region is located in... TRIM37 Genetically, the linkage between loci in this region was calculated using the linkage disequilibrium principle, and two highly adjacent and fully linked (LD=1) loci, rs431829386 and rs316872042, were identified as a pair of molecular genetic markers related to egg weight. Figure 2 They are all located in TRIM37 Gene.

[0017] 4) Determination of the relationship between genotype and egg weight of this molecular genetic marker pair: Based on this large population, the genotypes of this pair of molecular genetic markers and the corresponding individuals' 32-week egg production phenotype data were extracted. It was found that the molecular genetic marker pair in the population was mainly composed of the reference mutant AA+GG and the heterozygous mutation AG+GT. The mean egg weight at 32 weeks for AA+GG was 54.28g, and the mean egg weight at 32 weeks for AG+GT was 54.09g, and there was a statistically significant difference between the two groups (P<0.01). Therefore, individuals with the AA+GG genotype were determined to have relatively high egg weight, while individuals with the AG+GT genotype were determined to have relatively low egg weight. Figure 3 This pair of molecular genetic markers can be used to predict an individual's future egg weight trait, and can be applied to breeding work involving egg weight selection to accelerate the breeding process.

[0018] 2. Detection method for this molecular genetic marker pair 1) Blood DNA extraction: Obtain 0.1 mL of venous blood from the individual to be tested, extract DNA from the blood sample, and use it as a PCR amplification template.

[0019] 2) PCR amplification and quality control: Amplification is performed using the specific primers and DNA template of this invention. The amplification product should show a single band at 641bp when detected by agarose gel electrophoresis, which can be used for subsequent sequencing.

[0020] 3) Sequencing of amplified products: Sanger sequencing is performed on the amplified products to compare the two-site genotypes.

[0021] 4) Result determination: AA+GG genotype indicates a relatively high egg weight individual, while AG+GT genotype indicates a relatively low egg weight individual.

[0022] 3. Accuracy verification and economic benefit assessment of this molecular genetic marker pair Another batch of 60 individuals were tested for accuracy using the detection method of this invention. The results showed that the egg weight of the molecular genetic marker was significantly higher in AA+GG individuals than in AG+GT individuals, indicating that this invention is accurate and reliable in predicting egg weight.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. This invention has identified molecular genetic marker pairs related to egg weight and developed a detection method based on PCR. The dual-marker detection can achieve early and accurate prediction of egg weight traits, which is of great value for reducing breeding costs and accelerating the selection of superior strains. 2. The method established in this invention features dual-molecular genetic marker detection, improving accuracy and offering greater simplicity and cost-effectiveness compared to gene chip and resequencing methods. In terms of economic benefits, the detection method described in this invention costs approximately 20 yuan per sample, while gene chip and low-depth resequencing methods cost approximately 200 yuan per sample. This invention, while improving accuracy and simplicity, reduces the detection cost by (200-20) / 200*100%=90% compared to gene chip and resequencing methods. Furthermore, in breeding applications, taking a 10,000-chicken farm as an example, this detection method, based on gene frequency calculations, can early cull 2% of relatively low-egg-weight individuals. With a farming cost of approximately 150 yuan per chicken, this translates to a saving of 2%*10000*150=30,000 yuan, or 30,000 yuan per 10,000 chickens. Utilizing the genetic molecular marker detection method of this invention allows for early identification of chickens with high or low egg weight traits, thereby guiding subsequent breeding or egg production. Attached Figure Description

[0024] Figure 1 Based on the results of a genome-wide association study, statistical association tests were performed between population-wide genome-wide SNPs and 32-week-old egg weight. A high-signal region was found on chromosome 19, and this region was included as a candidate genetic marker. The outer ring in the figure indicates chromosome number and SNP density, while the area from the circumference to the center indicates -log10( P_wald The statistical value, that is, the closer to the center of the circle, the more significant it is. The yellow and red dashed lines indicate the significance threshold. Figure 2 The annotation and linkage disequilibrium of the molecular genetic marker pair involved in this invention refers to the case where the molecular genetic marker pair is physically close, fully linked, and simultaneously located in the TRIM37 gene; Figure 3 The data shows the egg weight corresponding to the molecular genetic marker pairs involved in this invention. It can be seen that the egg weight of individuals with the AA+GG genotype is significantly higher than that of individuals with the AG+GT genotype. Figure 4 This diagram illustrates the reading of base information using the dual-marker detection method of this invention. By comparing the peak diagrams at positions 201bp and 605bp of the reference sequence provided by this method, the genotype of the molecular genetic marker pair is read. If the 201bp peak is a homozygous single peak for A, it is determined to be the AA genotype; if it is a heterozygous double peak for AG, it is determined to be the AG genotype. If the 605bp peak is a homozygous single peak for G, it is determined to be the GG genotype; if it is a heterozygous double peak for GT, it is determined to be the GT genotype. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0026] Example 1: Screening and Validation of the Molecular Genetic Marker Pair 1) Acquisition of Population Egg Weight Phenotypic and Genotypic Data: This example uses 3450 Rhode Island Red hens. Egg weight data of this population at 32 weeks of age was recorded, and the statistical description of the data is shown in Table 1. Blood was collected from the subwing vein of this population, and DNA was extracted from each whole blood sample using a blood DNA extraction kit. Genotypic data of the population was obtained by detection using an SNP chip (“Jingxin No. 1”, Beijing Compson Agricultural Technology Co., Ltd.).

[0027] Table 1

[0028] 2) Genome-wide association analysis (GEMMA) was used to screen molecular marker pairs associated with egg weight: A mixed linear model (y = Xβ + Zu + ϵ, where y is the phenotypic vector, Xβ is the fixed effect, Zu is the random effect, and ϵ is the residual term) was employed in GEMMA v.0.98.5 software to perform statistical association tests between population genomic SNPs and 32-week-old egg weight. The results are as follows: Figure 1As shown, two sites were found that were physically close (400 bp), completely linked (LD=1), and located at the same TRIM37 site in the gene. Figure 2 As shown, these two sites were included in the candidate markers.

[0029] 3) Validation of the molecular genetic marker pair: In this population, 54 individuals with the genotype AG+GT for the molecular marker pair rs431829386 and rs316872042 had an average egg weight of 54.09g at 32 weeks of age, while 3396 individuals with the genotype AA+GG had an average egg weight of 54.28g at 32 weeks of age. This difference was statistically significant. P Genotypes with <0.01 (higher than AG+GT) such as Figure 3 As shown, this molecular genetic marker can effectively identify egg weight.

[0030] Example 2: Detection method and accuracy verification of this molecular genetic marker pair 1) Blood collection: Using blood collection needles and anticoagulant EDTA vacuum blood collection tubes, 0.1 mL of whole blood was collected from the subwing veins of 60 10-day-old Loch Ness Red hens and 60 10-day-old White Leghorn hens for subsequent testing.

[0031] 2) Blood DNA extraction: Blood DNA extraction kits were used to extract DNA from blood samples according to the operating procedures. The DNA concentration was measured and controlled at around 100 ng / μL for subsequent PCR.

[0032] 3) PCR reaction: Specific primers designed for this molecular genetic marker were used to perform PCR amplification with the extracted DNA (reagents are shown in Table 2). A 20 μL reaction system was used; the reaction system composition and procedure are shown in Tables 3 and 4. The amplification products were used for subsequent reactions.

[0033] Table 2 Table 3 Table 4

[0034] 4) Quality control and sequencing of amplified products: Take 1 μL of amplified product, mix with 1 μL of nucleic acid dye, and use a DNA marker. Perform 1.5% agarose gel electrophoresis in the same batch. After electrophoresis, if a single band of 641 bp is observed, the quality control is passed and the product can be used for subsequent sequencing.

[0035] 5) For the amplification products that pass quality control, Sanger sequencing is used to obtain sequencing data. This data is then compared with the reference sequence provided in this method (5'-3' (SEQ ID NO.3)). Base information at positions 201bp and 605bp is read using the following method: Figure 4 As shown.

[0036] 6) Result Interpretation: If both genotypes show AA+GG, the individual is considered to have a relatively high egg weight; if they show AG+GT, the individual is considered to have a relatively low egg weight. An example of interpretation is shown below. Figure 4 Individuals can be selected for elimination based on breeding and production goals.

[0037] 7) Verification: Egg weights of the two breeds of hens at 32 weeks of age were recorded. For the Loch Ness Red breed, 3 individuals (AG+GT) had an average egg weight of 53.59g at 32 weeks, and 57 individuals (AA+GG) had an average egg weight of 55.32g at 32 weeks. For the White Leghorn breed, 4 individuals (AG+GT) had an average egg weight of 52.44g at 32 weeks, and 56 individuals (AA+GG) had an average egg weight of 53.87g at 32 weeks. This indicates that the detection method can accurately identify individuals with different egg weights at an early stage.

Claims

1. The application of molecular genetic markers rs431829386 and rs316872042 in predicting egg weight trait, characterized in that, The molecular genetic markers rs431829386 and rs316872042 are located at positions 7311314 and 7311718 on chromosome 19, respectively.

2. The application of a detection kit containing molecular genetic markers rs431829386 and rs316872042 in predicting egg weight traits, characterized in that, The molecular genetic markers rs431829386 and rs316872042 are located at positions 7311314 and 7311718 on chromosome 19, respectively.

3. The application according to claim 1 or 2, characterized in that, The molecular genetic markers rs431829386 and rs316872042 are of AG+GT or AA+GG type.

4. The application according to claim 1 or 2, characterized in that, When the genotype is AA+GG, the individual has a relatively high egg weight; when the genotype is AG+GT, the individual has a relatively low egg weight.

5. A specific primer pair for molecular genetic markers rs431829386 and rs316872042, characterized in that, The nucleotide sequences of the specific primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The application of the specific primer pair of rs431829386 and rs316872042 described in claim 5 in the determination of egg weight trait, characterized in that, The nucleotide sequences of the specific primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.

2.

7. The application of a kit containing the specific primer pairs of the molecular genetic markers rs431829386 and rs316872042 as described in claim 5 in the determination of egg weight traits, characterized in that, The nucleotide sequences of the specific primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. The application according to claim 6 or 7, characterized in that, Includes the following steps: (1) The hen DNA was amplified by PCR to obtain the amplification product; the nucleotide sequences of the PCR amplification primers are shown in SEQ ID NO.1 and SEQ ID NO.2; (2) Agarose gel electrophoresis was performed, and the amplified product with a band size of 641 bp was sequenced and compared with the nucleotide sequence shown in SEQ ID NO.3; (3) When the genotype at rs431829386 is AA and the genotype at rs316872042 is GG, the tested individual is determined to be a relatively high egg weight individual; when the genotype at rs431829386 is AG and the genotype at rs316872042 is GT, the tested individual is determined to be a relatively low egg weight individual.

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