A molecular marker primer for the ELMO1 gene associated with egg production traits in chickens at 300 days of age and its application.

Abstract

This invention relates to a trait related to egg production in chickens at 300 days of age. ELMO1 This invention relates to gene molecular marker primers and their applications, belonging to the field of biotechnology. The molecular marker of this invention is located at base 46261915 on chromosome 2 of the chicken reference genome GRCg7b version, exhibiting T and C single nucleotide polymorphisms. Two genotypes, TT and TC, exist at this molecular marker location, with individuals of the TC genotype showing significantly higher egg production at 300 days of age than those of the TT genotype. This invention can be used to screen for high-producing, high-quality chicken individuals, improve the uniformity and overall level of egg production in flocks at 300 days of age, achieve early and precise selection of high-quality chickens, help accelerate the breeding process, improve breeding accuracy, and reduce breeding and production costs, possessing significant economic application value and breeding promotion value.

Description

Technical Field

[0001] This invention relates to a molecular marker primer for the ELMO1 gene, which is associated with egg production traits in chickens at 300 days of age, and its application, belonging to the field of biotechnology. Background Technology

[0002] With increasing demands for egg quality and laying performance, high-efficiency production of laying hens and dual-purpose breeds has become a crucial goal in poultry breeding. Laying performance is one of the core economic traits for measuring hen productivity. Among these, 300-day-old egg production comprehensively reflects a hen's laying potential throughout the pre-laying and peak periods, making it a commonly used and important evaluation indicator in breeding practice, and of great significance for the selection and breeding of laying hens and local breeds. Egg production is a typical quantitative trait in chickens, regulated by multiple genes and easily influenced by factors such as feeding management, nutritional levels, and environmental conditions. 300-day-old egg production is not only closely related to the hen's sexual maturity process and reproductive system development, but also directly affects economic benefits throughout the breeding cycle. In actual production, improving the overall level and uniformity of 300-day-old egg production in a flock helps stabilize peak laying and extend the high-efficiency laying period, thereby improving breeding efficiency.

[0003] Currently, selection of egg production in chickens at 300 days of age mainly relies on phenotypic records, i.e., evaluation is conducted through long-term feeding and continuous egg production statistics after chickens reach the corresponding age. However, this method has significant shortcomings: on the one hand, phenotypic testing is time-consuming and costly, requiring substantial investment in feed and management resources; on the other hand, selection can only be carried out after chickens reach 300 days of age, making it difficult to promptly cull low-producing individuals and reducing breeding efficiency. Furthermore, phenotypic selection is easily affected by environmental factors, resulting in limited accuracy and hindering early and precise selection of superior breeds. Molecular marker-assisted selection technology provides a new approach for poultry breeding. By screening functional genes or molecular markers significantly associated with egg production traits, egg production potential can be predicted in early stages of chicken development, thereby enabling early selection, improving breeding accuracy, and reducing breeding costs. However, stable molecular markers for egg production traits at 300 days of age in chickens are still relatively limited, especially molecular markers related to key regulatory genes and their applications, which lack systematic research.

[0004] Studies have shown that the ELMO1 (Engulfment and Cell Motility 1) gene is an important functional gene involved in cell migration, cytoskeleton remodeling, and phagocytosis, playing a crucial role in tissue development and physiological regulation. Related research suggests that cell migration and tissue remodeling are significant in reproductive physiology processes such as ovarian development and follicle growth, and the ELMO1 gene has an expression basis in reproductive-related tissues, potentially participating in reproductive function regulation. Since egg production at 300 days of age in chickens is influenced by multiple factors, including ovarian development and ovulation efficiency, it exhibits a complex genetic regulatory mechanism. However, current research on the relationship between the ELMO1 gene and egg production in chickens remains limited, and its application in marker-assisted selection for egg production performance lacks systematic reports. Therefore, exploring the genetic association between the ELMO1 gene and egg production at 300 days of age in chickens, and developing stable and effective molecular markers, is of great significance for enriching the molecular breeding theoretical basis of egg production traits and improving the early selection efficiency of egg production performance. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a molecular marker primer for the ELMO1 gene related to egg production traits in chickens at 300 days of age and its application, which can rapidly and accurately identify egg production traits in chickens at 300 days of age.

[0006] This invention determined the egg production of Chishui Silkie hens at 300 days of age, used whole-genome resequencing technology for SNP genotyping, and screened the ELMO1 gene molecular marker, which is significantly associated with the egg production trait at 300 days of age, through genome-wide association analysis. This provides new gene and molecular marker resources for molecular-assisted breeding of chickens with egg production traits.

[0007] This invention solves the technical problem through the following technical solution: First, it provides a molecular marker for the ELMO1 gene that is significantly associated with egg production in chickens at 300 days of age. This molecular marker is located at base 46261915 on chromosome 2 of the chicken reference genome GRCg7b version. The reference genome base is T, and the mutant base is C. The site exhibits T / C base polymorphism. This site is located in exon 1 of the ELMO1 gene (corresponding to transcript XM 046923634.1 of the NCBI gene accession number), belonging to the 5'UTR (untranslated region exons). The deoxyribonucleotide sequences of the chicken DNA-specific primer pair required for the detection of the molecular marker are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] The above-mentioned specific primer pairs were used for molecular detection of egg production traits in chickens at 300 days of age.

[0009] This invention further provides applications of the aforementioned specific primers, including the detection of SNP genotypes related to egg production traits in chickens at 300 days of age. Specifically, it applies to a method for detecting SNP genotypes related to egg production in chickens at 300 days of age using PCR amplification combined with Sanger sequencing. The detection method includes the following steps: The first step is to perform PCR amplification on the chicken DNA sample to be tested using chicken DNA-specific primers to obtain the amplification product. The chicken DNA sample to be tested contains the 46261915th base of chromosome 2 of the chicken reference genome GRCg7b version. The second step is to perform Sanger sequencing on the amplified products. The third step is to determine the molecular marker genotype of the target site based on the sequencing results from the second step.

[0010] In the first step of the above method, the deoxyribonucleotide sequence of the chicken DNA-specific primer pair is determined by... Upstream primer: 5'-CCAGGATACGGTTGGCTCTT-3' (SEQ ID NO:1) Downstream primer: 5'-CCTCTTCCTTCCTCCTGCTG-3' (SEQ ID NO:2) The amplification product is 458 bp in length and contains the 46261915th base on chicken chromosome 2.

[0011] The final concentration of the reaction system, calculated as 25 μl, is: 50 ng of chicken DNA template to be tested 2 x Rapid Taq Master Mix 12.5μl upstream primer 1 μl 1 μl of downstream primer Add ddH2O to a final volume of 25 μl. The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 3 min; then 35 cycles were performed, each cycle consisting of denaturation at 95°C for 15 sec, annealing at 57.5°C for 15 sec, extension at 72°C for 15 sec; and finally extension at 72°C for 5 min, followed by storage at 4°C.

[0012] The nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0013] In the third step, the criterion is that the egg production of chickens with the T / C genotype at the SNP site is higher than that of chickens with the T / T genotype at 300 days of age.

[0014] This invention utilizes the ELMO1 gene molecular marker to detect the genotype of egg production in chickens at 300 days of age. Results showed that individuals with the TC genotype had a higher egg production at 300 days of age than those with the TT genotype. Using chicken genomic DNA as a template, PCR amplification was performed using chicken-specific primer pairs. Sanger sequencing and SNP molecular marker genotyping of the amplified products allowed for molecular-assisted selection of the egg production trait at 300 days of age based on the genotype of the SNP locus.

[0015] In breeding practice, individuals with the TC genotype can be preferentially selected based on established breeding goals, thereby improving the overall egg production level and flock uniformity at 300 days of age. Its beneficial effects include: efficient and rapid identification of egg production performance at 300 days of age, enabling early selection of individuals with high egg production potential, and providing a scientific basis for molecular marker-assisted breeding of high-quality chickens. Furthermore, the detection method disclosed in this invention has a simple operating procedure, good reproducibility, and can be completed under routine molecular biology experimental conditions. It also has the potential for further application in large-scale genomic breeding and molecular-assisted selection technologies, demonstrating promising prospects for widespread application. Attached Figure Description

[0016] Figure 1 This is a Manhattan plot of GWAS analysis of egg production traits in chickens at 300 days of age.

[0017] Figure 2 This is a gel imaging image of the DNA amplification product at the target mutation site.

[0018] Figure 3 These are Sanger sequencing results of PCR amplification products from three genotypes.

[0019] Figure 4 This is a box plot showing the phenotypic distribution of individuals with two genotypes based on the chr2:46261915 molecular marker.

[0020] Figure 5 This is a graph showing the differences in ELMO1 gene expression in the liver, ovarian stroma, small white follicles, and small yellow follicles of chickens in different egg production groups. Detailed Implementation

[0021] The following examples are used for the breeding of Chishui Silkie chickens.

[0022] Example This embodiment uses Chishui Silkie hens (purchased in March 2022 from Luyuan Poultry Industry Co., Ltd., Luoba Village, Baiyi Township, Wudang District, Guiyang City, Guizhou Province) as the research subject. The cumulative egg production from the start of egg production to 300 days of age was recorded. SNP genotyping was performed using whole-genome resequencing technology. Genome-wide association analysis (GWAS) was used to screen for the ELMO1 gene molecular marker, which was significantly associated with egg production at 300 days of age. The results are as follows: Figure 1 As shown.

[0023] This embodiment uses the following experiments to identify and apply the molecular marker of the ELMO1 gene, which is related to the egg production trait at 300 days of age.

[0024] 1. Phenotyping and Genotyping (1) Phenotypic analysis of egg production in chickens at 300 days of age Two hundred and fifty Chishui Silkie hens were selected as experimental animals and raised under the same feeding conditions. Free access to feed and water was provided throughout the experiment. Egg production was continuously recorded for each hen from the start of laying, and the cumulative egg production at 300 days of age was calculated as phenotypic data for egg production at 300 days of age.

[0025] (2) Extraction of genomic DNA Blood was collected from the subwing vein of the chickens to be tested. After anticoagulation, 200 μL of blood was added to columns 1 and 7 of the TianGen DP605 kit according to the kit instructions, along with 20 μL of proteinase K. The 96-well plate was then placed on the TGuide S16 automated nucleic acid extraction and purification base. The automatic extraction program of the TGuide S16 automated nucleic acid extraction and purification instrument (Table 1) was run. Table 1

[0026] After the program finishes running, aseptically collect the DNA extract from column 5 of the deep well plate and store it at 4°C for later use.

[0027] (3) PCR amplification Using the extracted genomic DNA as a template, the fragment containing the SNP molecular marker at position 46261915 of chicken chromosome 2 was amplified.

[0028] Upstream primer: 5'-CCAGGATACGGTTGGCTCTT-3' (SEQ ID NO:1) Downstream primer: 5'-CCTCTTCCTTCCTCCTGCTG-3' (SEQ ID NO: 2) The final concentration of the reaction system (25 μl) is: DNA to be tested 50 ng 2 x Rapid TCq Master Mix 12.5 μl upstream primer 1 μl 1 μl of downstream primer Add ddH2O to a final volume of 25 μl. The PCR amplification reaction conditions were as follows: ① 95℃ pre-denaturation for 3 min; ② 95℃ denaturation for 15 sec; ③ 57.5℃ annealing for 15 sec; ④ 72℃ extension for 15 sec (then jump to ②, for a total of 35 cycles); ⑤ 72℃ extension for 5 min; storage at 4℃. 2 μl was used for agarose gel detection, and an amplification product with a single target band length of 458 bp was obtained (gel imaging as shown). Figure 2 (As shown) and contains base position 46261915 of chicken chromosome 2. The sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4: SEQ ID NO:3 CCAGGATACGGTTGGCTCTTCTTTTAAAAAGACAAGAAATTTTACTCCACCGCTTTTCCAGCGAGCGGCACCGCTGTCAAACCTCGGGGGCCGGCGGGGCGGGGCGGTGAGGGGCTCCCAGCCGTGCTACTGCCGCCCCCTCCCGGCCGGGCCGGGGGAGGCTCTGCTCGGGGTGCCCCGTGCCGGGCTCGGAGCTGCTCGCTAGGGTCGGGGCGTAGCGGTACGCGTG GCAAAGTGCCCGCCGCGGGGCCGAACGGAGGTGCGGGTTCTGCCGGTTAAAGGAATTAAACTAACCGTATTGTAAAGGCAGCGAGGACCGGTGGGAACTCTCCTCCGGAGCCGTGCGGGAGGAGGCACAGCCGTGACCGAAGAGAGGCGGCTGGGCCCGTCCGGTCCCCGCAGGTGCCGGAGCCCCGCGGGCTGCACGTCGCGGCCCGGCCAGCAGGAGGAAGGAAGAGG SEQ ID NO:4 CCAGGATACGGTTGGCTCTTCTTTTAAAAAGACAAGAAATTTTACTCCACCGCTTTTCCAGCGAGCGGCACCGCTGTCAAACCTCGGGGGCCGGCGGGGCGGGGCGGTGAGGGGCTCCCAGCCGTGCTACTGCCGCCCCCTCCCGGCCGGGCCGGGGGAGGCTCTGCTCGGGGTGCCCCGTGCCGGGCTCGGAGCTGCTCGCTAGGGTCGGGGCGTAGCGGTACGCGTG GCAAAGTGCCCGCCGCGGGGCCGAACGGAGGTGCGGGTTCTGCCGGTTAAAGGAATTAAACCAACCGTATTGTAAAGGCAGCGAGGACCGGTGGGAACTCTCCTCCGGAGCCGTGCGGGAGGAGGCACAGCCGTGACCGAAGAGAGGCGGCTGGGCCCGTCCGGTCCCCGCAGGTGCCGGAGCCCCGCGGGCTGCACGTCGCGGCCCGGCCAGCAGGAGGAAGGAAGAGG (4) Sequencing verification and genotyping The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram is shown below. Figure 3 As shown.

[0029] (5) Transcriptome sequencing and ELMO1 Gene expression analysis To analyze the differences between individuals with high and low egg production ELMO1 To investigate the differences in gene expression in different tissues, the 250 Chishui Silkie chickens were sorted from highest to lowest cumulative egg production at 300 days of age. The six chickens with the lowest egg production (low egg production group, sample numbers Sample2-Sample7) and the six chickens with the highest egg production (high egg production group, sample numbers Sample8-Sample13) were selected. After aseptic necropsy, four tissues were collected: liver, ovarian stroma, small white follicles, and small yellow follicles. Each tissue sample was approximately 100 mg and was immediately flash-frozen in liquid nitrogen, and then transferred to a -80℃ freezer for later storage.

[0030] ① Total RNA Extraction and Quality Assay: Total RNA was extracted from each of the tissue samples according to the TRIzol reagent (Invitrogen, USA) instructions. The concentration of the extracted RNA was determined using a NanoDrop 2000 micro spectrophotometer, and the purity was assessed by the OD260 / 280 ratio. RNA integrity (RIN value) was then detected using an Agilent 2100 Bioanalyzer. Only qualified samples with an OD260 / 280 ratio between 1.8 and 2.1 and a RIN ≥ 7.0 were selected for subsequent library construction.

[0031] ② Library Construction and Sequencing: 1 μg of total RNA from each qualified sample was used as the starting template. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads. The mRNA was then fragmented, reverse transcribed using random primers to synthesize the first-strand cDNA, synthesized the second-strand cDNA, repaired the ends, added A tails, ligated adapters, screened for fragment size, and amplified by PCR to construct a strand-specific sequencing library. After the library passed quality control, paired-end 150 bp sequencing (PE150) was performed on the BGI MGI high-throughput sequencing platform. The raw data was then converted into FASTQ format raw reads after base identification using WriteFQ software.

[0032] ③ Data Processing and Expression Calculation: The raw sequencing data (Raw Reads) underwent quality control, filtering the following three categories of reads sequentially to obtain high-quality clean reads: (i) adapter-contaminated reads, i.e., reads with adapter-contaminated bases greater than 5 bp; (ii) low-quality reads, i.e., reads with a quality value Q ≤ 19 accounting for more than 50% of the total bases; (iii) reads containing N proportion greater than 5%. For paired-end sequencing, if one end read was filtered, the other end read was also removed. The clean reads of each sample were aligned to the chicken reference genome (GRCg7b version) using HISAT2 software. The alignment results were used for transcript assembly and expression quantification using StringTie software. The expression level of each sample was calculated using the Transcripts Per Million (TPM) method. ELMO1 The expression levels of the gene in the liver, ovarian stroma, small white follicles, and small yellow follicles are shown in Table 3.

[0033] 2. Results Analysis Differences in egg production phenotypes were analyzed in 250 green-shelled egg-laying hens aged 300 days with clear phenotypic records. Statistical tests were performed using the t-test function in R4.0 software, and pairwise comparisons of means were used to statistically test the genotype and egg production traits at 300 days of age in the experimental flock. P< 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. Results are shown in Table 2 and... Figure 4 As shown, among the tested individuals, there were 174 T / T genotypes and 76 T / C genotypes, and the difference in egg production at 300 days of age between the two genotypes was highly significant. p < 0.01). The average egg production at 300 days of age was 87.974 eggs, significantly higher than the 68.425 eggs produced by individuals with the T / C genotype. p < 0.01). Individuals with the T / C genotype have a higher average egg production, and lower standard deviation and coefficient of variation, indicating that the egg production performance of this genotype is relatively stable and the population uniformity is high.

[0034] These results indicate that chickens ELMO1 Genetic molecular markers are significantly correlated with the egg production trait of chickens at 300 days of age. Depending on the actual breeding goals, individuals with the T / C genotype can be selected to increase the egg production of chickens at 300 days of age, or individuals with the TT genotype can be selected to decrease the egg production of chickens at 300 days of age, thereby regulating the overall uniformity of egg production in the flock at 300 days of age and thus improving breeding efficiency.

[0035] To investigate the different tissues in high and low egg production groups ELMO1 To investigate gene expression differences, this embodiment collected liver, ovarian stroma, small white follicles, and small yellow follicles from the low egg production group (n=6, samples 2-7) and the high egg production group (n=6, samples 8-13) for transcriptome sequencing to obtain... ELMO1 Gene expression level (TPM). The results showed ( Figure 5 In the liver, the low-production group ELMO1 The expression level (3.71±0.86, mean ± SD) was significantly higher than that in the high-yield group (2.67±0.52, ...). p < 0.05, indicating a significant difference between the two groups; however, in ovarian stroma (low-yielding 44.58±12.98 vs high-yielding 43.65±10.42), small white follicles (low-yielding 37.91±12.59 vs high-yielding 35.63±11.51), and small yellow follicles (low-yielding 25.94±4.51 vs high-yielding 25.88±8.54), there were no substantial differences in expression levels between the two groups. In summary, ELMO1 The gene showed expression differences related to egg production performance only in liver tissue, with upregulated expression in the low egg production group, suggesting that the gene may be involved in regulating egg production traits in the liver, and that high expression of the gene may inhibit follicle selection and development.

[0036] Table 2. Association analysis between the molecular marker at position 46261915 on chromosome 2 and egg production trait in chickens at 300 days of age.

[0037] Note: Data with the same subscript letter in the same column indicate no significant difference, while data with different subscript letters indicate a significant difference. P <0.05).

[0038] Table 3. Transcriptome sequencing ELMO1 Gene expression levels (TPM) in high and low laying hens

[0039] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. The application of primers for an ELMO1 gene SNP molecular marker associated with egg production traits in Chishui Silkie chickens at 300 days of age, characterized in that: The SNP molecular marker is located at base 46261915 on chromosome 2 of the chicken reference genome GRCg7b version, with a base mutation of T or C, resulting in genotypes T / T and T / C. The deoxyribonucleotide sequences of the SNP molecular marker primers are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The application is to detect the SNP molecular marker genotypes related to egg production traits in Chishui Silkie chickens at 300 days of age. Chishui Silkie chickens with the TC genotype have a higher egg production at 300 days of age than those with the TT genotype.

2. The application of the primers for the ELMO1 gene SNP molecular marker related to the egg production trait of Chishui Black-boned Chicken at 300 days of age as described in claim 1, characterized in that: The detection method includes the following steps: The first step is to perform PCR amplification on the extracted chicken DNA sample using the SNP molecular marker primers to obtain the amplification product. The second step is to perform Sanger sequencing on the amplification products obtained in the first step. The third step is to determine the SNP molecular marker genotype of the target site in the chicken individual to be tested based on the sequencing results of the second step. The target site is the 46261915th base on chromosome 2 of the chicken reference genome GRCg7b version.

3. The application of the primers for the ELMO1 gene SNP molecular marker related to the egg production trait of Chishui Black-boned Chicken at 300 days of age as described in claim 2, characterized in that: The amplified product is 458 bp in length.

4. The application of the primers for the ELMO1 gene SNP molecular marker related to the egg production trait of Chishui Black-boned Chicken at 300 days of age as described in claim 2, characterized in that: In the first step, the final concentration of the reaction system, expressed as 25 μl, is: 50 ng of chicken DNA template to be tested 2 x Rapid Taq Master Mix 12.5μl upstream primer 1 μl 1 μl of downstream primer Add ddH2O to a final volume of 25 μl. The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 3 min; then 35 cycles were performed, each cycle consisting of denaturation at 95°C for 15 sec, annealing at 57.5°C for 15 sec, extension at 72°C for 15 sec; and finally extension at 72°C for 5 min, followed by storage at 4°C.

5. The application of the primers for the ELMO1 gene SNP molecular marker related to the egg production trait of Chishui Black-boned Chicken at 300 days of age as described in claim 4, characterized in that: The nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4.

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