Application of mitochondrial molecular marker in improvement of genetic breeding of chicken first-laying day age

Abstract

The invention provides an application of a mitochondrial molecular marker in improvement of chicken first laying day age genetic breeding, and belongs to the technical field of molecular genetics and animal breeding, the mitochondrial molecular marker is located at the 2071th site of chicken mitochondrial DNA and corresponds to the 542bp site in the sequence shown in SEQ ID NO.1, and the basic group at the site is C or T. The invention further provides an application of the mitochondrial molecular marker in improvement of chicken first laying day age genetic breeding. The mitochondrial molecular marker is associated with the laying day age of hens, and when the mitochondrial molecular marker is applied to genetic breeding of the hens, the laying day age and the egg laying performance of the hens can be genetically improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics and animal breeding, and particularly relates to an application of a mitochondrial molecular marker in genetic breeding for improving the laying age of chickens. Background Art

[0002] When selecting poultry for egg production, key indicators such as egg number and age at first laying are often used for evaluation. Age at first laying is positively correlated with egg number, meaning that the earlier the age at first laying, the longer the peak egg production period. Age at first laying can generally be observed in chickens over 20 weeks of age, and selecting for egg production based on this indicator facilitates early selection. Therefore, age at first laying is a key indicator used for egg production selection in laying hen breeding practices.

[0003] The mitochondrial genome, the genetic material outside the nucleus of animal cells, is characterized by a simple structure, rapid evolution, maternal inheritance, and a low rate of genomic recombination. Mitochondrial genomes vary significantly among species, while also exhibiting some variability within a species. This variability can serve as an important characteristic for breed identification. Recent studies have demonstrated that mitochondria, as regulatory centers for energy metabolism, play a crucial role in regulating reproductive performance in poultry. However, few studies have examined the impact of genetic variation in mitochondrial DNA on economically important traits in poultry. Summary of the Invention

[0004] In order to obtain a new method for improving the age at which chickens start laying, the present invention provides an application of a mitochondrial molecular marker in genetic breeding for improving the age at which chickens start laying. The mitochondrial molecular marker is located in the mitochondrial 12S sequence and is associated with the age at which hens start laying. Applying it to genetic breeding of chickens can genetically improve the age at which chickens start laying and egg-laying performance.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides an application of a mitochondrial molecular marker in genetic breeding for improving the laying age of chickens. The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO.1, where the base is C or T.

[0007] Based on the same inventive concept, the present invention provides an early selection method for the trait of the age of laying chicken, the early selection method comprising performing early selection on the trait of the age of laying chicken based on the genotype of mitochondrial molecular markers;

[0008] The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO. 1, where the base is C or T.

[0009] Furthermore, the early selection method specifically includes:

[0010] detecting the genotype of the mitochondrial molecular marker in the mitochondrial genome of the chicken to be tested;

[0011] Performing early selection on the laying age trait of the chicken to be tested based on the genotype of the mitochondrial molecular marker;

[0012] Among them, the CC genotype group of the mitochondrial molecular marker has an earlier laying age than the TT genotype group, and the TT genotype group has an earlier laying age than the CT genotype group. In the early selection process, the CC and / or TT genotype groups are retained, and the CT genotype group is eliminated.

[0013] Furthermore, the detecting of the genotype of the mitochondrial molecular marker in the mitochondrial genome of the chicken to be tested specifically includes:

[0014] The mitochondrial genome of the chicken to be tested was amplified by PCR using forward primer F and reverse primer R;

[0015] Sequencing the PCR amplification product to obtain the genotype of the mitochondrial molecular marker;

[0016] The nucleotide sequence of the forward primer F is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer R is shown in SEQ ID NO.3.

[0017] Based on the same inventive concept, the present invention provides primers for detecting mitochondrial molecular markers, the primers comprising a forward primer F and a reverse primer R, the nucleotide sequence of the forward primer F being shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer R being shown in SEQ ID NO.3;

[0018] The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO. 1, where the base is C or T.

[0019] Based on the same inventive concept, the present invention provides the use of primers for detecting mitochondrial molecular markers in genetic breeding for improving the laying age of chickens.

[0020] Based on the same inventive concept, the present invention provides a kit comprising a forward primer F and a reverse primer R.

[0021] Based on the same inventive concept, the present invention provides the use of the above-mentioned kit in genetic breeding for improving the laying age of chickens.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. The present invention discloses an application of a mitochondrial molecular marker in genetic breeding for improving the laying age of chickens. The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, belonging to the 12S rRNA region of the chicken mitochondrial genome, where the base is C or T. The mitochondrial molecular marker is associated with the laying age of hens. Applying the marker to genetic breeding of chickens can genetically improve the laying age and egg-laying performance of chickens. Moreover, since the marker is located in the mitochondrial genome, the marker can be used to perform early breeding selection based on the laying age of hens from the parent generation, effectively reducing breeding costs.

[0024] 2. The present invention discloses an application of a mitochondrial molecular marker in genetic breeding for improving the age of chicken laying, and discovers a mitochondrial DNA molecular marker that affects the age of chicken laying. When the molecular marker is used for molecular marker-assisted selection, the operation is simpler and the reliability is good. Early selection can be achieved in the maternal generation, greatly reducing breeding costs. The present invention also provides identification primers for the molecular marker. Through the molecular marker and primer, the Sanger sequencing method can be used to establish a fast, efficient and accurate molecular marker-assisted breeding technology, which can achieve earlier selection for the age of laying and accelerate genetic progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is the reference peak diagram for the base mutation at position 2071 of the mitochondrial DNA gene. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0028] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] The overall idea of the present invention is as follows:

[0031] The production model of the modern poultry industry usually adopts a family production method. The age at which hens start laying and their genetic potential greatly affect the egg-laying performance of their offspring. By measuring the mitochondrial DNA of hens, the composition of the mitochondrial DNA of their offspring can be determined, thereby effectively reducing the breeding workload during production. The mitochondrial 12S sequence (12S rRNA) is a ribosomal RNA gene sequence with important functional and structural characteristics, which plays a key role in the correct assembly and maturation of ribosomes. The inventors have discovered that 12S rRNA has both a certain degree of conservation and variability during the evolutionary process, and can be used as a molecular variety tag for research. However, no studies have yet reported the correlation between the 12S rRNA sequence and the age at which chickens start laying. Therefore, the inventors believe that exploring molecular markers in the 12S rRNA sequence that can be used for the age at which chickens start laying will provide a cost-effective genetic improvement method for chicken breeding selection.

[0032] The inventors conducted a lot of practical exploration and screening of mitochondrial DNA sequences. By downloading the mitochondrial genome sequences of different chicken species from NCBI, they creatively discovered a molecular marker in the chicken mitochondrial genome. This molecular marker is located in the 12SrRNA region, specifically at position 2071 of the mitochondrial DNA (GenBank:: MZ911748.1), where the base is C or T. By designing PCR primers containing this position, blood was collected from the specialized egg-laying chicken lines of the unit and genomic DNA was extracted. After PCR amplification and sequencing, the detection and analysis found that the age of the homozygous genotype CC or TT group was significantly earlier than that of the heterozygous genotype CT group. The combination of this molecular marker and primer is used to determine the chickens with the CC or TT genotype for seed selection. The method is convenient and effective, and can be used to achieve early seed selection in the chicken breeding process, thereby achieving high-quality development of poultry breeding.

[0033] The following will describe in detail the application of a mitochondrial molecular marker in the genetic breeding of improved chicken laying age in combination with examples and experimental data.

[0034] Example 1

[0035] This embodiment provides an application of mitochondrial molecular markers in genetic breeding for improving the laying age of chickens, specifically including:

[0036] 1. Experimental materials, reagents and instruments

[0037] (1) Experimental materials: The fifth generation of green-shell laying hens from the Jiangsu Poultry Science Research Institute were used as the material. All the hens had their laying age recorded. 188 hens were randomly selected and their blood was collected.

[0038] (2) Main reagents: Proteinase K solution (Sangon Biotechnology); 2×Accurate Taq premix (containing dye, Acrylic Biologicals); agarose (Sangon Biotechnology); DNA marker (Acrylic Biologicals).

[0039] (3) Main instruments: PCR amplification instrument (Bio-Rad), gel imaging analysis system (WD-9413B, Beijing Liuyi).

[0040] 2. Detection Method

[0041] (1) Chicken blood collection: Blood was collected from the wing vein of the candidate chicken group and anticoagulated with ACD.

[0042] (2) Genomic DNA extraction: Take the chicken blood sample in step (1) and extract the genomic DNA using the phenol imidazole method to determine the DNA concentration and purity.

[0043] (3) PCR amplification and sequencing: PCR reaction was performed using the genomic DNA obtained in step (2) as a template. The primers were: F: 5'-GGGTTGGTAAATCTTGTGCC-3' (SEQ ID NO. 2), R: 5'-TGATGGCTTGTGAAGAGGGT-3' (SEQ ID NO. 3). The primer positions are shown in bold at the beginning and end of SEQ ID NO. 1.

[0044] The PCR reaction system consisted of 25 μL of 2× Accurate Taq Master Mix, 1 μL each of 10 μM primers F and R, 2 μL of DNA template (final concentration ≤ 500 ng), and sterile water to 50 μL. The PCR reaction program was as follows: 30 cycles of initial denaturation at 94°C for 30 seconds, denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute, followed by a post-extension at 72°C for 5 minutes.

[0045] The obtained PCR product was detected on agarose gel electrophoresis, and a clear amplified band of about 661 bp was obtained, which was the target band.

[0046] After confirming successful amplification, Sanger sequencing was performed: the correctly amplified PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. for bidirectional sequencing.

[0047] (4) Identify the target position mutation in the candidate individual's mitochondrial DNA sequence: Use SnapGene software to view the sequencing results in step (3) and compare them with the following Seq ID No: 1 sequence.

[0048] Seq ID No:1:

[0049]

[0050]

[0051] The mutation position is marked as position 542 in SEQ ID NO.1. C / T mark , the sequencing peak diagrams of different genotypes of CC, TT, and CT are as follows Figure 1 shown.

[0052] (5) The distribution of the first laying age of different genotypes at this locus in L3 laying hens was compared, and the results are shown in Table 1. As shown in Table 1, the CC and TT homozygous genotypes are dominant genotypes, with the first laying age of 157.40 and 157.46 days, respectively, which are significantly earlier than the CT heterozygous genotype by 3.92 and 3.86 days, respectively.

[0053] Table 1 Differences in the age of onset of ovulation among different genotypes with mutations at base 2071 in the mitochondrial 12S region

[0054]

[0055] Therefore, during the egg production selection breeding process, CC and TT genotypes are selected and CT genotype is eliminated.

[0056] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0057] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0058] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. An application of a mitochondrial molecular marker in genetic breeding for improving the laying age of chickens, characterized in that: The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO. 1, where the base is C or T.

2. A method for early selection of chicken laying age traits, characterized in that: The early selection method comprises performing early selection on the chicken laying age trait based on the genotype of mitochondrial molecular markers; The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO. 1, where the base is C or T.

3. The early selection method for chicken laying age traits according to claim 2, characterized in that: The early selection method specifically includes: detecting the genotype of the mitochondrial molecular marker in the mitochondrial genome of the chicken to be tested; Performing early selection on the laying age trait of the chicken to be tested based on the genotype of the mitochondrial molecular marker; Among them, the CC genotype group of the mitochondrial molecular marker has an earlier laying age than the TT genotype group, and the TT genotype group has an earlier laying age than the CT genotype group. In the early selection process, the CC and / or TT genotype groups are retained, and the CT genotype group is eliminated.

4. The early selection method for the trait of the first-laying age according to claim 3, characterized in that: The detecting the genotype of the mitochondrial molecular marker in the mitochondrial genome of the chicken to be tested specifically comprises: The mitochondrial genome of the chicken to be tested was amplified by PCR using forward primer F and reverse primer R; Sequencing the PCR amplification product to obtain the genotype of the mitochondrial molecular marker; The nucleotide sequence of the forward primer F is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer R is shown in SEQ ID NO.

3.

5. A primer for detecting mitochondrial molecular markers, characterized in that: The primers include a forward primer F and a reverse primer R, the nucleotide sequence of the forward primer F is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer R is shown in SEQ ID NO.3; The mitochondrial molecular marker is located at position 2071 of the chicken mitochondrial DNA, corresponding to position 542 bp in the sequence shown in SEQ ID NO. 1, where the base is C or T.

6. Use of the primer as claimed in claim 5 in genetic breeding for improving the age of chicken laying.

7. A kit, characterized in that The kit comprises the primers according to claim 5.

8. Use of the kit as claimed in claim 7 in genetic breeding for improving the laying age of chickens.

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