Method for identifying pure-bred liancheng white duck by molecular biology and application thereof

Abstract

The application discloses a pure-breed Liancheng white duck molecular biology identification method and application, the molecular marker utilizes the combination haplotype of SNP1 and SNP2 to distinguish two types of ducks; the NCBI database ID number of the reference duck mitochondrial genome standard sequence is NC009684, the SNP1 is located at 271bp of the duck mitochondrial genome standard sequence, has TC polymorphism, the SNP2 is located at 296bp of the duck mitochondrial genome standard sequence, has AG polymorphism; when the combination haplotype is CA, it is Liancheng white duck, when the combination haplotype is TA or CG, it is Liancheng white duck containing foreign bloodline hybridization.The molecular marker can quickly and accurately identify Liancheng white duck and Liancheng white duck containing foreign bloodline hybridization, and is simple in operation, easy to operate in a laboratory, convenient for grass-roots promotion and use, can produce considerable economic benefits and good social value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a molecular biological identification method and its application for purebred Liancheng white ducks. Background Technology

[0002] The Liancheng White Duck, also known as the White Duck, originated in Liancheng County, Longyan City, Fujian Province. It is a rare poultry breed that is as famous as the Silkie Chicken, and is valued for its meat, eggs, and medicinal properties.

[0003] The Liancheng White Duck is a small duck breed with a slow growth rate, reaching an adult weight of only 1.3-18 kg. In recent years, to develop and utilize its resources, the faster-growing Cherry Valley White-feathered Meat Duck has been introduced as a maternal lineage (hereinafter referred to as an introduced lineage) to improve the Liancheng White Duck. This has resulted in hybrid Liancheng White Ducks containing introduced lineage, which resemble the original Liancheng White Duck in appearance, grow faster, and offer better economic benefits. However, the original genetic resource of the Liancheng White Duck must be well protected to prevent the invasion of introduced lineages. Therefore, a rapid, accurate, and easy-to-operate technique is urgently needed to distinguish between Liancheng White Ducks and hybrid Liancheng White Ducks containing introduced lineages, ensuring the purity of the Liancheng White Duck's bloodline.

[0004] Traditional methods for variety identification primarily rely on morphological characteristics and phenotypic traits. However, these methods are often significantly influenced by environmental factors and struggle to accurately distinguish varieties with similar genetic backgrounds. With the development of molecular biology techniques, single nucleotide polymorphism (SNP) markers, as highly efficient genetic markers, are widely used in variety identification. Haplotype molecular marker technology detects SNPs in an individual's genome and combines multiple SNPs to differentiate between different varieties or individuals. It offers advantages such as ease of operation, objective and fair results, and high reliability. Summary of the Invention

[0005] One of the objectives of this invention is to provide a molecular biological identification method for purebred Liancheng white ducks, addressing the shortcomings of existing technologies.

[0006] A second objective of this invention is to provide primers for detecting the aforementioned molecular markers.

[0007] A third objective of this invention is to provide the use of the aforementioned molecular markers.

[0008] The fourth objective of this invention is to provide a detection method utilizing the aforementioned molecular markers.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a molecular marker for distinguishing between Liancheng White Duck and Liancheng White Duck with foreign bloodlines. The molecular marker uses a combination haplotype of SNP1 and SNP2 to differentiate between the two types of ducks. Referring to the NCBI database ID number NC009684 of the standard duck mitochondrial genome sequence, SNP1 is located at 271 bp of the standard duck mitochondrial genome sequence and has TC polymorphism, while SNP2 is located at 296 bp of the standard duck mitochondrial genome sequence and has AG polymorphism. When the combined haplotype is CA, it is Liancheng White Duck; when the combined haplotype is TA or CG, it is Liancheng White Duck with foreign bloodlines (as shown in Table 1).

[0010] Table 1. Haplotypes of different types of meat ducks

[0011] This invention also provides a method for identifying Liancheng White Ducks and Liancheng White Ducks with foreign bloodlines. The method involves extracting genomic DNA from the duck to be tested, amplifying it by PCR, and performing Sanger sequencing. Sequence alignment is then performed, referencing the NCBI database ID number NC009684 for the standard duck mitochondrial genome sequence. SNP1 is located at 271 bp of the standard duck mitochondrial genome sequence and exhibits TC polymorphism; SNP2 is located at 296 bp of the standard duck mitochondrial genome sequence and exhibits AG polymorphism. When the combined haplotype of SNP1 and SNP2 is CA, it indicates a Liancheng White Duck; when the combined haplotype of SNP1 and SNP2 is TA or CG, it indicates a Liancheng White Duck with foreign bloodlines.

[0012] This invention also provides a primer pair for identifying Liancheng White Duck and Liancheng White Duck containing foreign bloodline hybrids, wherein: Forward primer (SEQ ID NO.1): 5'-CGGACTACCCTCCCAATGG-3' Reverse primer (SEQ ID NO.2): 5'-CGCCAACCGTCTCATTGAG-3'.

[0013] The present invention also provides a kit containing the above primer pairs for identifying Liancheng White Duck and haplotypes of Liancheng White Duck containing foreign bloodline hybrids.

[0014] The present invention also provides the application of the above-mentioned molecular markers or primers or kits in the identification of Liancheng White Duck and Liancheng White Duck containing foreign bloodline hybrids.

[0015] The present invention also provides the application of the above-mentioned molecular markers or primers or kits in screening Liancheng White Ducks and Liancheng White Ducks containing foreign bloodlines.

[0016] This invention also provides a method for distinguishing between Liancheng White Duck and Liancheng White Duck hybrids containing foreign bloodlines. The method involves amplifying duck genomic DNA using primer pairs SEQ ID NO.1 / SEQ ID NO.2, performing Sanger sequencing, and then performing sequence alignment analysis. Referring to the NCBI database ID number NC009684 for the standard duck mitochondrial genome sequence, SNP1 is located at 271 bp of the standard duck mitochondrial genome sequence and exhibits TC polymorphism; SNP2 is located at 296 bp of the standard duck mitochondrial genome sequence and exhibits AG polymorphism. When the combined haplotype of SNP1 and SNP2 is CA, it is a Liancheng White Duck; when the combined haplotype of SNP1 and SNP2 is TA or CG, it is a Liancheng White Duck hybrid containing foreign bloodlines.

[0017] In one specific embodiment, the present invention also provides a rapid haplotype identification method for Liancheng White Duck and Liancheng White Duck containing foreign bloodline hybrids, comprising the following steps: 1) Extract genomic DNA from the ducks to be tested; 2) Using the DNA extracted in step 1) as a template (the above-mentioned Liancheng white duck and the Liancheng white duck containing foreign blood hybrids), PCR amplification reaction is performed using the primer pair described in this invention; 3) After the reaction, the PCR products were sequenced by Sanger and then haplotyped. The NCBI database ID number for the standard duck mitochondrial genome sequence is NC009684. SNP1 is located at 271 bp of the standard duck mitochondrial genome sequence and has TC polymorphism. SNP2 is located at 296 bp of the standard duck mitochondrial genome sequence and has AG polymorphism. When the combined haplotype of SNP1 and SNP2 is CA, it is Liancheng White Duck. When the combined haplotype of SNP1 and SNP2 is TA or CG, it is Liancheng White Duck with foreign bloodline hybrid.

[0018] The PCR reaction system in step 2) of this invention is as follows: 25 μL of 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.), 1 μL each of 10 μmol / L forward primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0019] The PCR reaction program in step 2) of this invention is as follows: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 7 min.

[0020] The genomic DNA of the Liancheng White Duck and the Liancheng White Duck with foreign bloodline to be detected in this invention can be extracted by common methods. The extraction site can be a site commonly used in the field, such as, but not limited to, duck meat, feathers or oral swabs. Beneficial effects

[0021] This invention utilizes haplotype identification to differentiate between Liancheng White Ducks and Liancheng White Ducks with foreign bloodlines. Its advantages include rapid and accurate identification of these two types. Furthermore, the method is simple to operate, easy to perform in laboratories, and convenient for grassroots promotion and use. In addition, the detection kit developed based on this method can generate considerable economic benefits and significant social value. Attached Figure Description

[0022] Figure 1 The CA haplotype sequencing diagram of the Liancheng white duck.

[0023] Figure 2 Sequencing image of CG haplotype of Liancheng White Duck, a hybrid species with foreign blood.

[0024] Figure 3 Sequencing data of the TA haplotype of the hybrid Liancheng White Duck containing foreign blood. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. Example

[0026] Identification of Liancheng White Duck and Liancheng White Duck with Foreign Bloodline Hybrids 1.1 Sample Collection Blood samples were collected from 30 Liancheng white ducks and 30 Liancheng white ducks with foreign bloodlines. Genomic DNA was extracted using a kit (Tiangen Biotech Co., Ltd.)

[0027] 1.2 PCR amplification PCR amplification was performed using primers designed according to this invention.

[0028] Forward primer: 5'-CGGACTACCCTCCCAATGG-3'; Reverse primer: 5'-CGCCAACCGTCTCATTGAG-3'.

[0029] The PCR reaction system is as follows: 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0030] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 7 min.

[0031] 1.3 Sanger sequencing After the reaction was completed, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0032] 1.4 Haplotype Statistics The distribution ratio of haplotypes in different groups is shown in the figure. Figures 1-3 and Table 2: Table 2. Haplotype distribution of Liancheng White Duck and Liancheng White Duck hybrids containing foreign bloodlines.

[0033] All 30 individuals of the Liancheng White Duck were haplotype CA, while 24 individuals of the hybrid Liancheng White Duck with foreign bloodlines were haplotype CG, and 6 individuals were haplotype TA. This demonstrates the high efficiency of this invention in identifying Liancheng White Ducks and Liancheng White Ducks with foreign bloodlines. Example

[0034] 2. Identifying genuine and fake "Liancheng White Duck" 2.1 Sample Collection Thirty-six slaughtered black-billed ducks that claimed to be "Liancheng White Ducks" were collected from the market, and genomic DNA was extracted using a kit (Tiangen Biotech Co., Ltd.).

[0035] 2.2 PCR amplification PCR amplification was performed using primers designed according to this invention.

[0036] Forward primer: 5'-CGGACTACCCTCCCAATGG-3'; Reverse primer: 5'-CGCCAACCGTCTCATTGAG-3'.

[0037] The PCR reaction system is as follows: 2×PCR Mix (Nanjing Novizan Biotechnology Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0038] The PCR reaction program was as follows: 95℃ for 5 min, (95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s) for 35 cycles, and 72℃ for 7 min.

[0039] 2.3 Sanger sequencing After the reaction was completed, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0040] 2.4 Haplotype Statistics Of the 36 ducks, 28 were of the CG haplotype, 8 were of the TA haplotype, and no CA haplotype was detected. The animal quarantine certificate issued by the seller was traced back to the source and confirmed that the ducks were of the Liancheng White Duck breed with foreign bloodlines.

[0041] In summary, this molecular marker is specific for Liancheng White Ducks and Liancheng White Ducks with foreign bloodlines. This also demonstrates the high efficiency of this invention in identifying Liancheng White Ducks and Liancheng White Ducks with foreign bloodlines.

Claims

1. The application of a substance for detecting molecular markers in distinguishing between purebred Liancheng White Ducks and hybrid Liancheng White Ducks with foreign maternal lineage; the molecular markers are the combined haplotypes of SNP1 and SNP2 to distinguish the two types of ducks; the NCBI database ID number for the standard duck mitochondrial genome sequence is NC009684, wherein SNP1 is located at 271 bp of the standard duck mitochondrial genome sequence and has T / C polymorphism, and SNP2 is located at 296 bp of the standard duck mitochondrial genome sequence and has A / G polymorphism; when the combined haplotype is CA, it is a purebred Liancheng White Duck, and when the combined haplotype is TA or CG, it is a hybrid Liancheng White Duck with foreign maternal lineage; the substance for detecting molecular markers is the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2 or a kit containing the primer pair.

2. A method for distinguishing between purebred Liancheng White Ducks and hybrid Liancheng White Ducks containing foreign maternal lineage, characterized in that, The duck genomic DNA was amplified by PCR, and after Sanger sequencing, sequence alignment analysis was performed. Referring to the NCBI database ID number NC009684 for the standard duck mitochondrial genome sequence, SNP1 was located at 271 bp of the standard sequence and exhibited T / C polymorphism, while SNP2 was located at 296 bp and exhibited A / G polymorphism. When the combined haplotype of SNP1 and SNP2 was CA, it indicated a purebred Liancheng White Duck; when the combined haplotype of SNP1 and SNP2 was TA or CG, it indicated a hybrid Liancheng White Duck containing foreign maternal lineage. The primer pairs used for amplification are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, The amplification reaction system was as follows: 25 μL of 2×PCR Mix, 1 μL each of the 10 μmol / L primers shown in SEQ ID NO.1 and SEQ ID NO.2, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

4. The method according to claim 2, characterized in that, The amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 7 min.

5. The method according to claim 2, characterized in that, The duck genomic DNA to be tested was obtained from duck meat, feathers, or oral swabs.

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