A lncRNA gene molecular marker related to chicken laying weight trait and its application
Through whole-genome resequencing technology, the lncRNA gene molecular markers related to weight production were screened out, and PCR amplification and Sanger sequencing were used to detect the weight production traits of chicken weight production, solving the problem of difficult to quickly identify the weight production of high-quality chickens in the existing technology. Efficient breeding and cost control have been achieved.
Patent Information
- Application Number
- CN202410870384.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-01
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2044-07-01
AI Technical Summary
It is difficult to quickly and accurately identify the weight traits of high-quality yellow-feathered broilers, resulting in high production costs and limited industrial development.
Through whole genome resequencing technology, lncRNA gene molecular markers related to weight production were screened out, and PCR amplification and Sanger sequencing were used for PCR amplification and Sanger sequencing were detected to detect SNP molecular markers at base 170764013 of Chicken chromosome No. 1, providing genotype information to guide breeding selection.
It has achieved rapid and accurate identification of the weight traits of chicken production, improved breeding efficiency and breeding efficiency, reduced waste of feeding costs, and provided a scientific basis for early breeding of high-quality chickens.
Smart Images
Figure CN118389713B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an IncRNA gene molecular marker related to the chicken's initial laying weight trait and application thereof, belonging to the field of biotechnology. Background Art
[0002] Single nucleotide polymorphism (SNP) refers to the variation of a single nucleotide in the genome. It can be stably inherited to offspring during the transmission of genetic material and is easy to detect. Therefore, it can be used as a molecular marker for population selection and seed conservation.
[0003] High-quality yellow-feathered broilers have better meat flavor, and are tolerant to roughage, resistant to diseases, and highly adaptable, making them more popular with consumers and breeders. The start of laying is an important signal that marks the sexual maturity of hens. Before sexual maturity, carcass weight increases with age and tends to level off after sexual maturity, and flavor substances accumulate; after sexual maturity, feed conversion rate decreases, pH value increases, inosinic acid and other contents decrease, crude protein content decreases, tenderness decreases, meat becomes older, and taste worse, so it is particularly important to choose the right age for listing. Yellow-feathered broilers have a long breeding cycle, slow growth rate, and late sexual maturity, resulting in high production costs, which has affected the development of the yellow-feathered broiler industry.
[0004] Sexual maturity traits of chickens include comb size, age at first laying, and weight at first laying, which are closely related to production performance and meat quality. Early sexual maturity is an important economic trait and selection index unique to high-quality yellow-feathered broilers. Generally speaking, as the weight at first laying increases, the age at first laying will increase, and it is positively correlated with the weight at first laying. Excessive or small weight at first laying will affect the quality of breeding eggs, thereby affecting breeding. Studies have found that when the growing chickens reach or exceed the standard weight, they can start laying in time; individuals with a weight at first laying below the standard weight may start laying late, the peak of egg laying will be postponed, and the egg production rate and survival rate will decrease. Chickens at an appropriate weight have the best egg-laying performance and a low feed-egg ratio. Therefore, breeding excellent weight at first laying traits can improve the performance of broiler breeders. However, the weight at first laying can only be measured at first laying, and selecting at first laying will cause a huge waste of feeding costs. Therefore, it is urgent to find suitable molecular markers to quickly and accurately identify the weight performance of high-quality chickens at first laying, so that early selection can be achieved and the purpose of uniform weight at first laying can be achieved.
[0005] Long non-coding RNA (lncRNA) is a type of long non-coding RNA with a length greater than 200 nt. It is an important component of the non-coding genome and is mainly involved in the control of chromatin structure, regulating enhancer activity, and participating in the formation of biomolecular condensates. Studies have found that lncRNA ENSGALG00000053256 is an important candidate gene affecting chicken body weight. Summary of the invention
[0006] The purpose of the present invention is to address the defects of the prior art and to propose a lncRNA gene molecular marker related to the chicken's initial laying weight trait and its application, which can quickly and accurately identify the chicken's initial laying weight trait.
[0007] The present invention measured the initial laying weight of yellow-feathered broiler hens, performed SNP genotyping using whole genome resequencing technology, and screened the lncRNAENSGALG00000053256 gene molecular marker significantly correlated with initial laying weight through whole genome association analysis, providing new gene and molecular marker resources for the breeding of chicken initial laying weight traits.
[0008] The present invention solves the technical problem through the following technical scheme: providing an IncRNA gene molecular marker related to the weight of chickens at the beginning of laying, wherein the molecular marker is located at the 170764013th base of chromosome 1 of chicken, the base mutation is C or T, the genotype is C / C, C / T and T / T, and the sequence is the 264th base shown in SEQ ID NO:3 or SEQ ID NO:4; the deoxyribonucleotide sequence of the chicken DNA specific primer pair required for molecular marker detection is shown in SEQ ID NO:1 and SEQ ID NO:2.
[0009] The above specific primer pairs were used for molecular detection of the trait of chicken laying weight.
[0010] The present invention further provides an application of IncRNA gene molecular markers related to the trait of chicken weight at the beginning of laying, including the use for detecting SNP genotypes related to the trait of chicken weight at the beginning of laying. Specifically applied to a method for detecting SNP genotypes related to the trait of chicken weight at the beginning of laying by PCR amplification combined with Sanger sequencing, the detection method comprises the following steps:
[0011] The first step is to perform PCR amplification on the chicken DNA sample to be tested using a chicken DNA specific primer pair to obtain an amplified product, wherein the chicken DNA sample to be tested contains a SNP molecular marker at the 170764013th base of chicken chromosome 1;
[0012] The second step is to perform Sanger sequencing on the amplified product;
[0013] The third step is to determine the molecular marker genotype of the target site based on the sequencing results of the second step.
[0014] In the first step of the above method, the deoxyribonucleotide sequence of the chicken DNA specific primer pair is
[0015] Upstream primer: 5'-TCCGACGCTTCTCATTCTCT-3' (SEQ ID NO: 1)
[0016] The downstream primer is composed of 5'-AACTACTGCCACAGGGAGGA-3' (SEQ ID NO: 2). The amplified product is 473 bp in length and includes the 170764013th base on chicken chromosome 1.
[0017] The final concentration of the reaction system is calculated based on 25 μl:
[0018] Chicken DNA to be tested 50 ng
[0019] 2 x Accurate Taq Master Mix 12.5μl
[0020] Upstream primer 1 μl
[0021] Downstream primer 1 μl
[0022] Add sterile water to 25 μl.
[0023] The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C.
[0024] The nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4.
[0025] In the third step, the judgment standard is that the first-laying weight of chickens with T / T genotype at the SNP site is higher than that of chickens with C / T genotype and C / C genotype, and the first-laying weight of chickens with C / T genotype is higher than that of chickens with C / C genotype.
[0026] The present invention detects the genotype of the chicken's first-laying weight trait through IncRNA gene molecular markers, and finds that the T / T genotype chicken's first-laying weight is higher than that of the C / T genotype chicken and the C / C genotype chicken, and the C / T genotype chicken's first-laying weight is higher than that of the C / C genotype chicken. By using the genomic DNA of the chicken to be tested as a template, using a specific primer pair for PCR amplification, and then performing Sanger sequencing and SNP molecular marker genotyping on the PCR amplification product, the genotype of the SNP molecular marker can be used to select the chicken's first-laying weight trait. In breeding, according to the breeding goal, by eliminating C / T genotype individuals and C / C genotype individuals, retaining T / T genotype individuals, the beneficial effect is that the chicken's first-laying weight trait can be identified efficiently and quickly, providing a scientific basis for the early selection and breeding of high-quality chickens. In addition, the detection method disclosed in the present invention is simple and easy to operate, and can be carried out in the laboratory. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the Manhattan plot of the GWAS analysis of birth weight traits.
[0028] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the three genotypes.
[0029] Figure 3 This is a box plot of the phenotypic distribution of individuals with three genotypes of the chr1:170764013 molecular marker. DETAILED DESCRIPTION
[0030] The following embodiments are applicable to the breeding of yellow-feathered broiler chickens.
[0031] Example
[0032] In this example, the weight of yellow-feathered broiler Suqin No. 3 L-line hens at the time of laying was measured, and SNP genotyping was performed using whole genome resequencing technology. The lncRNA ENSGALG00000053256 gene molecular marker significantly associated with the weight at the time of laying was screened through whole genome association analysis. The results are as follows: Figure 1 shown.
[0033] In this example, the following experiments were conducted to identify and apply IncRNA gene molecular markers related to the chicken's initial laying weight trait.
[0034] 1. Phenotypic and genotypic testing
[0035] (1) Experimental materials and determination of birth weight phenotype
[0036] 630 hens of the yellow-feathered broiler Suqin No. 3 L line were selected as experimental animals and raised under the same feeding conditions, with free access to food and water throughout the process. At the beginning of laying, the laying weight of each chicken was recorded in turn as the phenotypic data of the chicken's laying weight.
[0037] (2) Extraction of genomic DNA
[0038] Blood was collected from the subwing vein of the individual to be tested, lysed after anticoagulation, digested with proteinase K, extracted with saturated sodium chloride method, dissolved in TE and stored at -20℃.
[0039] (3) PCR amplification
[0040] Using the genomic DNA extracted as above as a template, a fragment containing the SNP molecular marker at base position 170764013 of chromosome 1 was amplified.
[0041] Upstream primer: 5'-TCCGACGCTTCTCATTCTCT-3' (SEQ ID NO: 1)
[0042] Downstream primer: 5'-AACTACTGCCACAGGGAGGA-3' (SEQ ID NO: 2)
[0043] The final concentration of the reaction system (25 μl) is:
[0044] DNA to be tested 50 ng
[0045] 2 x Accurate Taq Master Mix 12.5 μl
[0046] Upstream primer 1 μl
[0047] Downstream primer 1 μl
[0048] Add sterile water to 25 μl.
[0049] The reaction conditions of PCR amplification are: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30sec, 60℃ annealing for 30sec, 72℃ extension for 60sec, 30 cycles in total; 72℃ extension for 2min; 20℃ storage; 10μl is taken for agarose detection, and a single target band with a length of 473bp is amplified, and the SNP molecular marker containing the 170764013th base of chicken chromosome 1 is obtained. The nucleotide sequence of the amplified product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.
[0050] (4) Sequencing verification and genotyping
[0051] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak graph was as follows: Figure 2 shown.
[0052] 2. Results Analysis
[0053] 630 Suqin No. 3 L strain hens with clear records of the phenotype of the first-laying weight were selected, and the R 4.2 ANOVA test function was used for statistical testing and drawing. The pairwise mean comparison mode was selected to perform statistical tests on the genotype and first-laying weight traits of the experimental chickens. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. The results are shown in Tables 1 and Figure 3As shown in the data, among the tested individuals, there were 46 C / C genotypes, 239 C / T genotypes, and 285 T / T genotypes. The differences in the first-laying weight of the three genotypes were extremely significant (P<0.01). The average first-laying weight of the T / T genotype individuals was 1727.65g, which was 98g higher than that of the C / T genotype individuals (P<0.01) and 186g higher than that of the C / C genotype individuals (P<0.01). The average first-laying weight of the C / T genotype individuals was 1629.04g, which was 130g higher than that of the C / C genotype individuals (P<0.01). The results showed that the chicken IncRNA gene molecular marker was significantly correlated with the first-laying weight trait of chickens. According to the actual breeding goals, T / T genotype individuals can be selected to improve the first-laying weight of high-quality broiler breeders, improve the overall uniformity of first-laying weight, and improve the breeding efficiency.
[0054] Table 1. Association analysis between the molecular marker at base 170764013 on chromosome 1 and the trait of weight at the beginning of calving
[0055] genotype Number of individuals Weight at birth / g CV C / C 46 <![CDATA[1542.41±175.73 a ]]> 11.39% C / T 239 <![CDATA[1629.04±175.56 b ]]> 10.78% T / T 285 <![CDATA[1727.65±175.24 c ]]> 10.14%
[0056] Note: Data in the same column with the same letters on their shoulders indicate no significant difference, while data with different letters on their shoulders indicate significant difference (P<0.05).
[0057] In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.
Claims
1. An application of lncRNA gene molecular markers related to the trait of chicken laying weight, characterized by: Used for molecular detection of the weight trait related to the start of laying of yellow-feathered broiler Suqin No. 3, the molecular marker is located at the 170764013th base of chromosome 1 of the chicken reference genome GRCg6a version, the base mutation is C or T, and the genotype is C / C, C / T and T / T; the deoxyribonucleotide sequence of the chicken DNA specific primer pair required for molecular marker detection is shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 3 or SEQ ID NO:
4.
2. The use of the lncRNA gene molecular marker related to the chicken laying weight trait according to claim 1, characterized in that: The detection method comprises the following steps, The first step is to perform PCR amplification on the chicken DNA sample to be tested using a chicken DNA specific primer pair to obtain an amplified product, wherein the chicken DNA sample to be tested contains a SNP molecular marker at the 170764013th base of chicken chromosome 1; The second step is to perform Sanger sequencing on the amplified product; The third step is to determine the lncRNA gene molecular marker genotype of the target site based on the sequencing results of the second step.
3. The use of the lncRNA gene molecular marker related to the chicken laying weight trait according to claim 2, characterized in that: The deoxyribonucleotide sequence of the chicken DNA-specific primer pair in the first step consists of an upstream primer of SEQ ID NO: 1 and a downstream primer of SEQ ID NO:
2. The amplified product is 473 bp in length and includes the 170764013th base on chicken chromosome 1.
4. The use of the lncRNA gene molecular marker related to the chicken laying weight trait according to claim 3, characterized in that: In the first step, the final concentration of the reaction system is calculated as follows based on 25 μl: Chicken DNA to be tested 50 ng 2 x Accurate Taq Master Mix 12.5μl Upstream primer 1 μl Downstream primer 1 μl Sterile water is added to make up to 25 μl. The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C.
5. The use of the lncRNA gene molecular marker related to the chicken laying weight trait according to claim 2, characterized in that: In the third step, the judgment standard is that the first-laying weight of chickens with T / T genotype at the SNP site is higher than that of chickens with C / T genotype and C / C genotype, and the first-laying weight of chickens with C / T genotype is higher than that of chickens with C / C genotype.