Application of a mitochondrial molecular marker in genetic breeding of improving chicken age at first egg

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics and animal breeding technology, and specifically relates to the application of a mitochondrial molecular marker in the genetic breeding of improved chicken laying age. Background Technology

[0002] When selecting poultry for egg production, important indicators such as the number of eggs laid and the age at first egg are usually used for evaluation. The age at first egg production is positively correlated with the number of eggs laid; that is, the earlier the age at first egg production, the longer the peak egg production period lasts. The age at first egg production can generally be observed in chickens around 20 weeks of age. Using this indicator to select for egg production facilitates early selection. Therefore, in egg-laying hen breeding practice, the age at first egg production is a key indicator used for selecting egg production.

[0003] The mitochondrial genome is the extranuclear genetic material of animal cells, characterized by its simple structure, rapid evolution, maternal inheritance, and low recombination rate. Mitochondrial genomes exhibit significant differences between species, and even within the same species, there is a degree of variability, which can serve as an important breed marker. Recent studies have shown that mitochondria, as the regulatory center of energy metabolism, play a crucial regulatory role in the control of poultry reproductive performance. However, research on the impact of genetic variation in mitochondrial DNA on important economic traits in poultry is scarce. Summary of the Invention

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

[0005] This invention is achieved through the following technical solution:

[0006] This invention provides the application of mitochondrial molecular markers in genetic breeding to improve the age of first laying in chickens. The mitochondrial molecular markers are located at position 2071 of 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 age at first laying trait in chickens, the early selection method comprising early selection of the age at first laying trait in chickens based on genotypes 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] Detect the genotype of the mitochondrial molecular markers in the mitochondrial genome of the chicken to be tested;

[0011] Early selection of the age at first laying trait in chickens based on the genotype of the mitochondrial molecular markers;

[0012] In this process, the age of onset of the CC genotype population with mitochondrial molecular markers is earlier than that of the TT genotype population, and the age of onset of the TT genotype population is earlier than that of the CT genotype population. In the early selection process, the CC and / or TT genotype populations are retained, while the CT genotype population is eliminated.

[0013] Furthermore, the detection 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] The PCR amplification products were sequenced 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, this invention provides the application of primers for detecting mitochondrial molecular markers in genetic breeding to improve the age of first laying in 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 application of the above-mentioned reagent kit in genetic breeding for improving the age of chickens to start laying.

[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 relates to the application of a mitochondrial molecular marker in the genetic breeding of improved chicken laying age. The mitochondrial molecular marker is located at position 2071 of chicken mitochondrial DNA, belonging to the 12S rRNA region of the chicken mitochondrial genome, where the base is C or T. This mitochondrial molecular marker is associated with the laying age of hens. Applying it to chicken genetic breeding can genetically improve the laying age and egg production performance of chickens. Furthermore, since the molecular marker is located in the mitochondrial genome, the application of this molecular marker allows for early breeding selection of the laying age of hens from the parent generation, effectively reducing breeding costs.

[0024] 2. This invention relates to the application of mitochondrial molecular markers in genetic breeding for improving the age of egg production in chickens. It identifies mitochondrial DNA molecular markers that influence the age of egg production in chickens. Using these molecular markers for marker-assisted selection simplifies the process, improves reliability, and allows for early selection in the maternal generation, significantly reducing breeding costs. This invention also provides primers for identifying these molecular markers. Using these markers and primers, a rapid, efficient, and accurate marker-assisted breeding technique can be established using Sanger sequencing, enabling earlier selection of the age of egg production and accelerating genetic progress. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0027] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0028] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning 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 pertains. In the event of any conflict, this specification shall prevail.

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

[0030] The overall concept of this invention is as follows:

[0031] Modern poultry farming typically employs family-based production methods, where the age at which hens begin laying and their genetic potential significantly influence the egg production performance of their offspring. By measuring the mitochondrial DNA of hens, the composition of the offspring's mitochondrial DNA can be determined, effectively reducing breeding workload in production. The mitochondrial 12S sequence (12S rRNA) is a ribosomal RNA gene sequence with important functional and structural features, playing a crucial role in the correct assembly and maturation of ribosomes. The inventors discovered that 12S rRNA exhibits both conservation and variability during evolution, making it suitable for use as a molecular breed marker. However, no studies have yet reported a correlation between the 12S rRNA sequence and the age at which chickens begin laying. Therefore, the inventors believe that exploring molecular markers within the 12S rRNA sequence that can be used for determining the age at which chickens begin laying will provide a cost-effective and efficient genetic improvement method for chicken breeding selection.

[0032] The inventors conducted extensive practical exploration and screening of mitochondrial DNA sequences. By downloading mitochondrial genome sequences of different chicken breeds from NCBI, they creatively discovered a molecular marker in the chicken mitochondrial genome located in the 12S rRNA 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, genomic DNA was extracted from blood samples collected from specialized laying hen breeds in their unit. After PCR amplification and sequencing, the results showed that the age of first egg production in homozygous CC or TT genotype groups was significantly earlier than that in heterozygous CT genotype groups. Using this molecular marker and primer combination to identify chickens with the CC or TT genotype for breeding is a convenient and effective method that enables early selection during chicken breeding, achieving high-quality development in poultry breeding.

[0033] The following will provide a detailed description of the application of a mitochondrial molecular marker in the genetic breeding of improved chicken laying age, in conjunction with embodiments and experimental data.

[0034] Example 1

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

[0036] 1. Experimental materials, reagents and instruments

[0037] (1) Experimental materials: The L3 fifth generation of green-shelled egg chickens from Jiangsu Provincial Institute of Poultry Science was used as the material. All chickens had records of their age at first laying. 188 hens were randomly selected and their blood was collected.

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

[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 veins of the candidate chicken population and anticoagulated with ACD.

[0042] (2) Genomic DNA extraction: Take the chicken blood sample from step (1) and extract genomic DNA using the phenol-formaldehyde 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 the bolded sequences 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 a final volume of 50 μL. The PCR reaction program was as follows: 94℃ pre-denaturation for 30 sec, 98℃ denaturation for 10 sec, 58℃ annealing for 30 sec, 72℃ extension for 1 min, for a total of 30 cycles, followed by a final extension at 72℃ for 5 min.

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

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

[0047] (4) Identify the mutation status of the target position in the mitochondrial DNA sequence of the candidate individual: 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 location is marked as position 542 in SEQ ID NO.1. C / T mark Sequencing peak diagrams of different genotypes of CC, TT, and CT are shown below. Figure 1 As shown.

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

[0053] Table 1. Differences in age at birth among different genotypes with mutations at nucleotide 2071 in the mitochondrial 12S region.

[0054]

[0055] Therefore, in selective breeding for egg production, the CC and TT genotypes are retained, while the CT genotype is eliminated.

[0056] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises 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 preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0058] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a mitochondrial molecular marker detection reagent in the genetic breeding of improved chickens' age at first laying, characterized in that, The mitochondrial molecular marker has a C or T base at position 542 bp in the sequence shown in SEQ ID NO. 1; The genotypes of the mitochondrial molecular markers in the genome of the chicken to be tested are detected. The laying age of the chicken population with the CC and TT genotypes of the mitochondrial molecular markers is earlier than that of the chicken population with the CT genotype. In the early selection process, the CC and / or TT genotype chicken populations are retained, and the CT genotype chicken populations are culled. The chicken breed is the L3 green-shelled egg-laying chicken strain.

2. The application according to claim 1, characterized in that, The detection reagent for the mitochondrial molecular marker includes primers for detecting the mitochondrial molecular marker, the primers including 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.

3. A method for early selection of chickens based on age at first laying, characterized in that, The early selection method includes early selection of chickens based on genotypes using mitochondrial molecular markers to determine the age at first laying. The mitochondrial molecular marker has a C or T base at position 542 bp in the sequence shown in SEQ ID NO. 1; The genotypes of the mitochondrial molecular markers in the genome of the chicken to be tested are detected. The laying age of the chicken population with the CC and TT genotypes of the mitochondrial molecular markers is earlier than that of the chicken population with the CT genotype. In the early selection process, the CC and / or TT genotype chicken populations are retained, and the CT genotype chicken populations are culled. The chicken breed is the L3 green-shelled egg-laying chicken strain.

4. The method according to claim 3, characterized in that, The genotypes of the mitochondrial molecular markers in the chicken genome to be tested were detected using the following steps: The genome of the chicken to be tested was amplified by PCR using forward primer F and reverse primer R; The PCR amplification products were sequenced 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.

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