DGKA gene molecular marker related to egg-laying cluster number and egg-laying persistence of laying hens, application and method

Abstract

The invention relates to the technical field of poultry genetic breeding and biology, in particular to a DGKA gene molecular marker for laying hen egg-laying persistent breeding, application and a method, and belongs to the technical field of molecular marker-assisted breeding. The molecular marker is located on a No.34 chromosome of a chicken reference genome bGalGal1. Mat.broil.GRCg7b, the 1334260th site has G / A polymorphism and is located in a 5 '-UTR region of a DGKA gene, and the SNP can be used as the molecular marker and is used for early genetic breeding of high-egg-yield persistent laying hens. The molecular marker is applied to early screening and genetic breeding of high-egg-yield persistent laying hens, breeding accuracy and breeding efficiency can be remarkably improved, phenotype determination cost is reduced, and the molecular marker has important economic value and popularization and application prospects for promoting efficient and precise development of the laying hen industry.

Description

Technical Field

[0001] This invention relates to the fields of poultry genetics and breeding and biotechnology, and particularly to a method related to the number of egg clusters and the sustainability of egg production in laying hens. DGKA Gene molecular markers, applications and methods. Background Technology

[0002] Egg production sustainability in laying hens represents a new requirement for breeding due to the demands of long-cycle laying hen farming (over 85 weeks). Egg production sustainability primarily focuses on traits such as the number of eggs laid and eggshell quality in the later stages of laying, reflecting a balanced breeding approach that considers both hen health and performance. Traditional breeding methods rely heavily on phenotypic data related to egg production performance for selection. This model has significant limitations: the breeding cycle is lengthy, requiring waiting for hens to enter the later stages of laying and accumulate sufficient data; feeding and testing costs are high; and it cannot accurately assess the genetic potential of breeder hens in early stages of growth and development (such as chicks or the growth period), leading to low selection efficiency. Our team's research found a significant correlation between the number of egg clusters and egg production sustainability in laying hens. Among individuals with the same number of eggs laid, those with more egg clusters exhibited better bone strength and eggshell quality, thus verifying that the number of egg clusters is an important trait for selective breeding of egg production sustainability in laying hens.

[0003] To overcome the bottlenecks of traditional breeding, molecular marker-assisted selection (MMR) technology has emerged. This technology analyzes DNA molecular markers closely linked to target traits, enabling precise determination of genetic advantages early in an individual's life, even before hatching. This allows for early and rapid screening of superior germplasm, significantly improving breeding efficiency and shortening the breeding cycle. However, the core prerequisite for this technology's application lies in discovering stable and reliable molecular markers that are significantly associated with important economic traits.

[0004] The diacylglycerol kinase family is a key regulator of intracellular signal transduction, participating in the regulation of various cellular functions by modulating lipid metabolism and second messenger pathways. Although the functions of DGK family members in mammals have been extensively studied, their biological roles in poultry, particularly laying hens, remain unclear. Currently, research on... DGKA Functional studies of the (diacylglycerol kinase α) gene in laying hens remain lacking, especially regarding its genetic association with the important economic trait of egg production persistence, which has not yet been reported. Therefore, there is an urgent need in this field to discover effective molecular markers closely related to egg cluster number and egg production persistence in laying hens, in order to solve the technical challenges currently faced in breeding practices and promote the advancement of laying hen breeding technology. Summary of the Invention

[0005] This invention aims to provide novel molecular markers significantly associated with egg cluster number and egg production sustainability in laying hens, addressing the lack of effective selection markers in existing technologies, and providing an early, rapid, and accurate method for selecting egg cluster number and egg production sustainability. To obtain molecular markers applicable to the selection of high egg cluster number and egg production sustainability in laying hens, the inventors, based on whole-genome resequencing data from laying hen populations with different egg cluster numbers, combined with SnpEff functional annotation, screened for markers located in... DGKA The SNP site g.1334260G>A in the 5'-UTR of the gene. Population validation revealed that this site is significantly correlated with the number of egg clusters: individuals carrying the A allele had a significantly higher average number of egg clusters than G / G homozygous individuals. This invention provides this SNP as a functional molecular marker for the early identification and precise breeding of laying hens with high egg cluster counts.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a molecular marker associated with the number of egg clusters and the persistence of egg production in laying hens. The molecular marker is nucleotide 1334260 on chromosome 34 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b. This site has G / A polymorphism and three genotypes: G / G, G / A, and A / A.

[0007] The present invention provides a primer pair for detecting the molecular marker, the sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] The present invention provides a kit for detecting the molecular marker, the kit comprising the primer pair described above.

[0009] Preferably, the kit further includes reagents for PCR amplification reactions.

[0010] This invention provides a method for detecting the number of egg clusters and molecular marker genotypes related to persistent egg production in laying hens, comprising the following steps: (1) Extract genomic DNA from the laying hens to be tested; (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification was performed using the primer pair to obtain an amplification product containing nucleotide 1334260 of chromosome 34 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b; (3) Sequencing or typing the amplification products obtained in step (2) to determine the genotype.

[0011] Preferably, the final volume of the PCR reaction system in step (2) is 20 μL, comprising: 50 ng template DNA, 10 μL 2×Accurate Taq Master Mix, 0.8 μL upstream primer, 0.8 μL downstream primer, and the remainder being dd... O; PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ extension for 2 min.

[0012] Preferably, in step (3), the genotype is determined by Sanger sequencing or competitive allele-specific PCR typing.

[0013] This invention provides the use of the molecular marker, the primer pair, or the kit in any of the following: (a) Preparation of products for continuous assisted breeding of laying hens; (b) Early genetic selection of laying hens; (c) Select individual laying hens with sustained high egg production or establish a core breeding group.

[0014] Preferably, the application includes: detecting the genotype of nucleotide 1334260 in the laying hen genome, and identifying individuals carrying the A allele as individuals with sustained high egg production potential.

[0015] Based on the above-described invention, the beneficial effects of the present invention are mainly reflected in the following two aspects: First, the SNP molecular marker located in the 5'-UTR region of the DGKA gene provided by this invention is significantly associated with the number of egg clusters and egg production persistence in laying hens, especially the carriers of the A allele, who exhibit a higher number of egg clusters. The discovery of this marker fills a gap in the genetic association study of the DGKA gene in laying hens, providing a reliable molecular target for early genetic selection of egg production persistence, and overcoming the technical bottlenecks of traditional breeding methods that rely on later phenotypic data, have long cycles, and are costly.

[0016] Secondly, this molecular marker and its associated detection methods can accurately identify individuals with sustained high egg production potential in the early stages of egg-laying hen growth and development. This not only significantly improves the accuracy and efficiency of breeding and shortens the breeding cycle, but also significantly reduces feeding and phenotyping costs. It has significant economic value and broad application prospects for promoting the development of the egg-laying hen industry towards efficient and precise breeding. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1

[0019] 1. Materials

[0020] Blood samples were collected from 114 Los Angeles Red chickens that showed no significant difference in egg production but significant difference in the number of egg clusters. The samples were stored at -80°C for genomic DNA extraction.

[0021] 2. Methods

[0022] 2.1 DNA Extraction and Quality Testing

[0023] Genomic DNA was extracted from whole blood using the phenol-chloroform method. The concentration (≥20 ng / μL) and purity (OD260 / 280=1.8~2.0) were detected by NanoDrop2000, and the integrity was confirmed by 1.5% agarose gel electrophoresis.

[0024] 2.2 SNP Site Screening and Annotation

[0025] Based on resequencing data, SnpEff (v5.1) was used to perform functional annotation of variants and screen for variants located in... DGKA Non-synonymous or regulatory SNPs within a ±10kb region of a gene.

[0026] 2.3 Genotyping and Phenotypic Association Analysis

[0027] Specific primers (SEQ ID NO: 1, 2) were designed targeting the g.1334260G>A site. Genotypes were determined by Sanger sequencing after PCR amplification. Fisher's exact test was used to analyze the association between genotype and egg cluster number. P <0.05 is considered significant.

[0028] 2.4 Primer Design

[0029] Primers were designed based on the chicken reference genome bGalGal1.mat.broiler.GRCg7b (chr34:1334160–1334360): Upstream primer (SEQ ID NO:1): 5′-TGGCTGCTGTTCTTCTGCTC-3′ Downstream primer (SEQ ID NO:2): 5′-CAGGACAGGGAGGAGAAGGA-3′ 3. Results Analysis Of the 114 valid samples, 20 laying hens carried the AA allele, 45 carried the GA allele, and 49 carried the GG allele. Fisher test P =0.017, indicating that the A allele is significantly positively correlated with the number of high-producing egg clusters.

[0030] Table 1. Association analysis between genotype and successive birth at the chr34:1334260 locus

[0031] This study shows that g.1334260G>A can serve as a functional molecular marker for the DGKA gene region, and can be used for molecular-assisted selection of laying hens with high egg production clusters and sustained egg production.

[0032] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker associated with the number of egg clusters and the persistence of egg production in laying hens, characterized in that, The molecular marker is nucleotide 1334260 on chromosome 34 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b. This site has a G / A polymorphism, with three genotypes: G / G, G / A, and A / A.

2. A primer pair for detecting the molecular marker of claim 1, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO:1 and SEQ ID NO:

2.

3. A kit for detecting the molecular marker of claim 1, characterized in that, The kit contains the primer pair as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also contains reagents for PCR amplification reactions.

5. A method for detecting the number of egg clusters and molecular marker genotypes related to egg production persistence in laying hens, characterized in that, Includes the following steps: (1) Extract genomic DNA from the laying hens to be tested; (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification was performed using the primer pair described in claim 2 to obtain an amplification product containing nucleotide 1334260 of chromosome 34 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b; (3) Sequencing or typing the amplification products obtained in step (2) to determine the genotype.

6. The method according to claim 5, characterized in that, In step (2), the final volume of the PCR reaction system is 20 μL, containing: 50 ng template DNA, 10 μL 2×Accurate Taq Master Mix, 0.8 μL upstream primer, 0.8 μL downstream primer, and the remainder is dd O; PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ extension for 2 min.

7. The method according to claim 5 or 6, characterized in that, In step (3), the genotype is determined by Sanger sequencing or competitive allele-specific PCR typing.

8. The use of the molecular marker of claim 1, the primer pair of claim 2, or the kit of claim 3 or 4 in any of the following: (a) To prepare products for continuous assisted breeding of laying hens; (b) Early genetic selection of laying hens; (c) Select individuals with high and sustained egg production or establish a core breeding group.

9. The application according to claim 8, characterized in that, The application includes: detecting the genotype of nucleotide 1334260 in the laying hen genome, and identifying individuals carrying the A allele as individuals with the potential for sustained high egg production.

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