Molecular biological identification method for purebred Liancheng white ducks and application

Abstract

The invention discloses a molecular biological identification method for purebred Liancheng white ducks and application. According to a molecular marker, two types of ducks are distinguished by utilizing a combined haplotype of SNP1 and SNP2; the ID number of an NCBI database of a reference duck mitochondrial genome standard sequence is NC009684, the SNP1 is located at the 271bp position of the duck mitochondrial genome standard sequence and has TC polymorphism, and the SNP2 is located at the 296bp position of the duck mitochondrial genome standard sequence and has AG polymorphism; when the combined haplotype is CA, the duck is Liancheng white duck, and when the combined haplotype is TA or CG, the duck is Liancheng white duck containing foreign consanguinity hybrid. The molecular marker can quickly and accurately identify Liancheng white ducks and Liancheng white ducks containing foreign consanguinity hybrids, is simple to operate, easy to operate in laboratories and convenient to popularize and use in grassroots, and can generate considerable economic benefits and good social value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a molecular biology identification method for purebred Liancheng white ducks and an application thereof. Background Art

[0002] Liancheng White Duck, also known as Baiwu Duck, is native to Liancheng County, Longyan City, Fujian Province. It is a rare poultry breed, on par with the Silkie chicken, used for its meat, eggs, and medicinal properties. According to the Liancheng County Chronicles, during the Daoguang reign of the Qing Dynasty, Tong Rongnan, in his book on the county's customs, noted that "ducks come in black and white varieties, with white ducks being considered the most beautiful and effective in treating dryness and heat. For those with a grand family feast, white ducks with black beaks are considered the most prized." The duck was listed as a "precious delicacy and tribute."

[0003] Liancheng White Duck is a small duck breed with a slow growth rate and an adult weight of only 1.3 to 18 kg. In recent years, for development and utilization, the faster-growing Cherry Valley White-feathered meat duck has been introduced as the maternal bloodline (hereinafter referred to as foreign bloodline) to improve the Liancheng White Duck. The hybrid Liancheng White Duck with foreign bloodline is similar in appearance to the Liancheng White Duck, grows faster, and has good economic benefits. But at the same time, the original germplasm resource of Liancheng White Duck must be well protected to avoid invasion by foreign bloodline. Therefore, there is an urgent need for a fast, accurate and easy-to-use technology to distinguish Liancheng White Duck from hybrid Liancheng White Duck with foreign bloodline to ensure the purity of the Liancheng White Duck bloodline.

[0004] Traditional breed identification methods rely primarily on morphological and phenotypic characteristics, but these methods are often significantly affected by environmental factors and struggle to accurately distinguish breeds with similar genetic backgrounds. With the development of molecular biology techniques, single nucleotide polymorphism (SNP) markers have become widely used as efficient genetic markers for breed identification. Haplotype molecular marker technology distinguishes different breeds or individuals by detecting SNPs in the genome of individual organisms and combining multiple SNPs. It offers advantages such as ease of use, objective and reliable identification results, and high reliability. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a molecular biological identification method for purebred Liancheng white duck in response to the shortcomings of the existing technology.

[0006] A second object of the present invention is to provide primers for detecting the above-mentioned molecular markers.

[0007] A third object of the present invention is to provide uses of the above-mentioned molecular markers.

[0008] A fourth object of the present invention is to provide a detection method using the above-mentioned molecular markers.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a molecular marker for distinguishing Liancheng white ducks and Liancheng white ducks with foreign bloodline. The molecular marker uses a combined haplotype of SNP1 and SNP2 to distinguish the 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 and has a TC polymorphism, and the SNP2 is located at 296bp of the duck mitochondrial genome standard sequence and has an AG polymorphism; when the combined haplotype is CA, it is a Liancheng white duck, and when the combined haplotype is TA or CG, it is a Liancheng white duck with foreign bloodline (as shown in Table 1).

[0011] Table 1 Haplotypes of different types of meat ducks

[0012]

[0013] The present invention also provides a method for distinguishing Liancheng white ducks and Liancheng white ducks with foreign bloodline. The method comprises the following steps: extracting genomic DNA of a duck to be detected, amplifying the duck by PCR, and performing Sanger sequencing, and then performing sequence comparison. The NCBI database ID number of the reference duck mitochondrial genome standard sequence is NC009684. SNP1 is located at 271 bp of the duck mitochondrial genome standard sequence and has a TC polymorphism. SNP2 is located at 296 bp of the duck mitochondrial genome standard sequence and has an AG polymorphism. When the combined haplotype of SNP1 and SNP2 is CA, the duck is a Liancheng white duck. When the combined haplotype of SNP1 and SNP2 is TA or CG, the duck is a Liancheng white duck with foreign bloodline.

[0014] The present invention also provides a primer pair for distinguishing a Liancheng white duck from a hybrid Liancheng white duck containing foreign blood, wherein:

[0015] Forward primer (SEQ ID NO. 1): 5'-CGGACTACCCTCCCAATGG-3'

[0016] Reverse primer (SEQ ID NO. 2): 5'-CGCCAACCGTCTCATTGAG-3'.

[0017] The present invention also provides a kit containing the primer pair for identifying the haplotypes of Liancheng white ducks and hybrid Liancheng white ducks containing foreign blood.

[0018] The present invention also provides application of the molecular markers, primers or kits in identifying Liancheng white ducks and hybrid Liancheng white ducks containing foreign blood.

[0019] The present invention also provides the use of the molecular markers, primers or kits in screening Liancheng white ducks and hybrid Liancheng white ducks containing foreign blood.

[0020] The present invention also provides a method for distinguishing Liancheng white ducks and Liancheng white ducks with foreign bloodline. The method comprises the following steps: amplifying duck genomic DNA using primer pair SEQ ID NO.1 / SEQ ID NO.2, performing Sanger sequencing, and performing sequence alignment analysis. With reference to the duck mitochondrial genome standard sequence, the NCBI database ID number of which is NC009684, SNP1 is located at 271 bp of the duck mitochondrial genome standard sequence and has a TC polymorphism. SNP2 is located at 296 bp of the duck mitochondrial genome standard sequence and has an AG polymorphism. When the combined haplotype of SNP1 and SNP2 is CA, the duck is a Liancheng white duck. When the combined haplotype of SNP1 and SNP2 is TA or CG, the duck is a Liancheng white duck with foreign bloodline.

[0021] In a specific embodiment, the present invention also provides a method for rapid identification of haplotypes of Liancheng White Duck and hybrid Liancheng White Duck containing foreign blood, comprising the following steps:

[0022] 1) Extracting genomic DNA from the duck to be tested;

[0023] 2) using the DNA extracted in step 1) as a template (the aforementioned Liancheng white duck and the hybrid Liancheng white duck containing foreign blood), and performing a PCR amplification reaction using the primer pair of the present invention;

[0024] 3) After the reaction, the PCR product was Sanger sequenced and haplotyped; the NCBI database ID number of the reference duck mitochondrial genome standard sequence is NC009684, SNP1 is located at 271bp of the duck mitochondrial genome standard sequence and has a TC polymorphism, and SNP2 is located at 296bp of the duck mitochondrial genome standard sequence and has an AG polymorphism; when the combined haplotype of SNP1 and SNP2 is CA, it is a Liancheng White Duck, and when the combined haplotype of SNP1 and SNP2 is TA or CG, it is a Liancheng White Duck containing foreign blood.

[0025] The PCR reaction system in step 2) of the present invention is: 25 μL of 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 1 μL of each 10 μmol / L forward primer, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0026] The PCR reaction program in step 2) of the present invention is: 95°C for 5 min, 35 cycles of (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), and 72°C for 7 min.

[0027] The genomic DNA of the Liancheng white duck to be tested and the hybrid Liancheng white duck containing foreign blood can be extracted by common methods, and the extraction location can be a location commonly used in the art, such as but not limited to duck meat, feathers or oral swabs.

[0028] Beneficial effects:

[0029] This method utilizes haplotypes to distinguish Liancheng white ducks from hybrids containing foreign bloodlines. Advantages include rapid and accurate identification of Liancheng white ducks from hybrids containing foreign bloodlines. Furthermore, the method is simple to operate, easy to perform in the laboratory, and readily accessible for grassroots promotion and use. Furthermore, the detection kit developed based on this method can generate substantial economic benefits and generate positive social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Sequencing map of Liancheng white duck CA haplotype.

[0031] Figure 2 Sequencing map of the CG haplotype of Liancheng White Duck, a hybrid with foreign bloodline.

[0032] Figure 3 TA haplotype sequencing map of Liancheng White Duck, a hybrid with foreign bloodline. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present 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.

[0034] Example 1

[0035] 1. Identification of Liancheng White Duck and its hybrids containing foreign blood

[0036] 1.1 Sample collection

[0037] Blood samples were collected from 30 Liancheng white ducks and 30 hybrid Liancheng white ducks containing foreign blood, and genomic DNA was extracted using a kit (Tiangen Biological Co., Ltd.).

[0038] 1.2 PCR amplification

[0039] The primers designed by the present invention were used for PCR amplification.

[0040] Forward primer: 5′-CGGACTACCCTCCCAATGG-3′;

[0041] Reverse primer: 5′-CGCCAACCGTCTCATTGAG-3′.

[0042] The PCR reaction system is:

[0043] 2×PCR Mix (Nanjing Novozymes Biotech Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, and ultrapure water 21 μL.

[0044] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), and 72°C for 7 min.

[0045] 1.3 Sanger sequencing

[0046] After the reaction, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0047] 1.4 Haplotype statistics

[0048] The haplotype distribution ratios of different populations are shown in Figure 1-Figure 3 And Table 2:

[0049] Table 2 Haplotype distribution of Liancheng White Duck and hybrid Liancheng White Duck with foreign bloodline

[0050]

[0051] All 30 Liancheng white duck haplotypes were CA, 24 of the hybrid Liancheng white ducks with foreign bloodline were CG haplotypes, and 6 were TA haplotypes. This also proves the high efficiency of the present invention in identifying Liancheng white ducks and hybrid Liancheng white ducks with foreign bloodline.

[0052] Example 2

[0053] 2. Identification of authentic "Liancheng White Duck"

[0054] 2.1 Sample collection

[0055] Thirty-six black-billed ducks, which were slaughtered in the market and called "Liancheng White Ducks", were collected and their genomic DNA was extracted using a kit (Tiangen Biological Co., Ltd.).

[0056] 2.2 PCR amplification

[0057] The primers designed by the present invention were used for PCR amplification.

[0058] Forward primer: 5′-CGGACTACCCTCCCAATGG-3′;

[0059] Reverse primer: 5′-CGCCAACCGTCTCATTGAG-3′.

[0060] The PCR reaction system is:

[0061] 2×PCR Mix (Nanjing Novozymes Biotech Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, and ultrapure water 21 μL.

[0062] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), and 72°C for 7 min.

[0063] 2.3 Sanger sequencing

[0064] After the reaction, the PCR products were sequenced by Sanger sequencing on an ABI 3730 sequencer.

[0065] 2.4 Haplotype statistics

[0066] Among the 36 ducks, 28 were CG haplotype, 8 were TA haplotype, and CA haplotype was not detected. After tracing the merchant's animal quarantine certificate, it was confirmed that the ducks were of the Liancheng white duck breed with foreign bloodline.

[0067] In summary, it can be seen that the molecular marker is specific for Liancheng White Duck and hybrid Liancheng White Duck with foreign bloodline, and also proves the high efficiency of the present invention in identifying Liancheng White Duck and hybrid Liancheng White Duck with foreign bloodline.

Claims

1. A molecular marker for distinguishing Liancheng White Duck from hybrid Liancheng White Duck containing foreign blood, characterized in that: The molecular marker utilizes the combined 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 and has a TC polymorphism, and the SNP2 is located at 296bp of the duck mitochondrial genome standard sequence and has an AG polymorphism; when the combined haplotype is CA, it is a Liancheng white duck, and when the combined haplotype is TA or CG, it is a Liancheng white duck containing a hybrid of foreign blood.

2. A primer pair for distinguishing a molecular marker of Liancheng white duck and a hybrid Liancheng white duck containing foreign blood, the sequence of which is as shown in SEQID NO.1 and SEQ ID NO.

2.

3. A kit comprising the primer pair according to claim 2.

4. Use of the molecular marker according to claim 1, the primer pair according to claim 2 or the kit according to claim 3 in distinguishing Liancheng white ducks and hybrid Liancheng white ducks containing foreign blood.

5. Use of the molecular marker according to claim 1, the primer pair according to claim 2 or the kit according to claim 3 in screening Liancheng white ducks and hybrid Liancheng white ducks containing foreign bloodlines.

6. A method for distinguishing Liancheng White Duck from hybrid Liancheng White Duck containing foreign blood, characterized in that: The genomic DNA of the duck to be tested was amplified by PCR, and sequence comparison analysis was performed after Sanger sequencing. The NCBI database ID number of the reference duck mitochondrial genome standard sequence was NC009684. SNP1 was located at 271bp of the duck mitochondrial genome standard sequence and had TC polymorphism. SNP2 was located at 296bp of the duck mitochondrial genome standard sequence and had AG polymorphism. When the combined haplotype of SNP1 and SNP2 was CA, it was a Liancheng white duck. When the combined haplotype of SNP1 and SNP2 was TA or CG, it was a Liancheng white duck containing foreign blood.

7. The method according to claim 6, characterized in that The primer pair used for amplification is shown in SEQ ID NO.1 and SEQ ID NO.

2.

8. The method according to claim 6, characterized in that The amplification reaction system was as follows: 25 μL of 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 1 μL of each 10 μmol / L forward primer, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

9. The method according to claim 6, characterized in that The amplification reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), and 72°C for 7 min.

10. The method according to claim 6, characterized in that The duck genomic DNA to be tested was obtained from duck meat, feathers or oral swabs.

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