Use of an indel marker, primers and detection kit in screening and / or identifying muscovy ducks

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to an application of an Indel marker, a primer and a detection kit in screening and / or identifying Muscovy duck. The application provides an Indel marker, which is located in the 2nd intron of a duck RASEF gene, specifically, a "TTGAGTT" fragment insertion or deletion at 11096bp-11102bp of the gene; the marker can be used to accurately identify Muscovy duck; the application further provides corresponding primers, detection kits and methods for specifically identifying Muscovy duck species. Experiments prove that, by using the technical scheme provided by the application, Muscovy duck can be specifically screened and / or identified from Beijing duck, Obaixing duck, Cherry Valley duck, Liancheng white duck, Xuixian curly duck, Jingjiang curly duck, Shanma duck, Shaoxing duck, Jinyun curly duck, Mianyang curly duck and Jinding duck, and the result is accurate, simple and fast.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of an Indel marker, primer, and detection kit in the screening and / or identification of Muscovy ducks. Background Technology

[0002] Muscovy ducks are excellent meat ducks, with good meat quality, low fat, and rich in vitamins, minerals, riboflavin, thiamine, and other nutrients. They have the effects of supplementing the body's nutrition, enhancing the body's immunity, and nourishing yin and cooling blood.

[0003] In the duckling market, impure breeds and inferior products are frequently sold as superior ones. Current technology typically identifies Muscovy ducks based on their appearance and behavior, but this process is not only cumbersome but also inaccurate. Furthermore, when duck blood, tissue, or DNA samples are mixed up, identification based on appearance and behavior becomes impossible, leading to unclear and uncertain duck species and seriously affecting research on related duck species. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Indel markers, primers, and detection kits in screening and / or identifying Muscovy ducks, which can accurately and quickly screen and identify Muscovy ducks.

[0005] This invention provides an Indel marker located in the second intron of the duck RASEF gene, specifically at positions 11096bp to 11102bp of the duck RASEF gene, where there is an insertion or deletion of the "TTGAGTT" fragment; the Gene ID of the duck RASEF gene is 101803543.

[0006] Preferably, the nucleotide sequence containing the Indel tag is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] This invention provides a method for amplifying the Indel marker described in the above-mentioned technical solution, wherein the primers include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3; and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0008] This invention provides a detection kit containing the primers and PCR amplification reagents described in the above technical solution.

[0009] Preferably, the PCR amplification reagents include DNA polymerase, dNTPs, and Mg. 2+ .

[0010] This invention provides the application of the Indel markers, primers, or detection kits described in the above technical solution in the screening and / or identification of Muscovy ducks.

[0011] This invention provides a method for screening and / or identifying Muscovy ducks, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain PCR amplification products; sequencing the PCR amplification products and making a judgment; when the sequence of the PCR amplification product contains the insertion of the "TTGAGTT" fragment, the sample to be tested is determined not to be a Muscovy duck; when the sequence of the PCR amplification product does not contain the "TTGAGTT" fragment, the sample to be tested is determined to be a Muscovy duck.

[0012] Preferably, the sample to be tested comes from one or more sources, including blood, tissue, and cells.

[0013] Preferably, the PCR amplification reaction system, in 25 μL, comprises: 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L upstream primer, 0.5 μL of 10 μmol / L downstream primer, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O.

[0014] Preferably, the PCR amplification reaction program is as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0015] Beneficial effects:

[0016] This invention provides an Indel marker located in the second intron of the duck RASEF gene, specifically at positions 11096bp to 11102bp, where an insertion or deletion of the "TTGAGTT" fragment is present. The Gene ID of the duck RASEF gene is 101803543. This marker enables accurate identification of Muscovy ducks. Furthermore, this invention provides primers and a detection kit for amplifying the aforementioned Indel marker to specifically identify Muscovy duck species.

[0017] This invention provides a method for screening and / or identifying Muscovy ducks, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain PCR amplification products; sequencing the PCR amplification products and making a judgment; when the sequence of the PCR amplification product contains the insertion of the "TTGAGTT" fragment, the sample to be tested is determined not to be a Muscovy duck; when the sequence of the PCR amplification product does not contain the "TTGAGTT" fragment, the sample to be tested is determined to be a Muscovy duck. Experiments have shown that, using the technical solution provided by this invention, Muscovy ducks can be specifically screened from Beijing ducks, Aobaixing ducks, Cherry Valley ducks, Liancheng white ducks, Youxian Muscovy ducks, Jingjiang Muscovy ducks, Mountain Muscovy ducks, Shaoxing ducks, Jinyun Muscovy ducks, Mianyang Muscovy ducks, and Jinding ducks, with accurate, simple, and rapid results. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The sequencing peak of the Beijing duck sequence in Example 1 Figure 1 ;

[0020] Figure 2 The sequencing peak of the Beijing duck sequence in Example 1 Figure 2 ;

[0021] Figure 3 The sequencing peak of the Muscovy duck sequence in Example 1 Figure 1 ;

[0022] Figure 4 The sequencing peak of the Muscovy duck sequence in Example 1 Figure 2 ;

[0023] Figure 5 This is a sequencing peak diagram of the Cherry Valley duck sequence from Example 2;

[0024] Figure 6 This is a sequencing peak diagram of the Muscovy duck sequence from Example 3;

[0025] Figure 7 This is a sequencing peak diagram of the Muscovy duck sequence from Example 4;

[0026] Figure 8 This is a sequencing peak diagram of the Yangxin Muscovy duck sequence from Example 5. Detailed Implementation

[0027] This invention provides an Indel marker located in the second intron of the duck RASEF gene, specifically at positions 11096bp to 11102bp, where an insertion or deletion of the "TTGAGTT" fragment is present; the Gene ID of the duck RASEF gene is 101803543. Compared to sequences in non-Muscovy duck breeds, Muscovy ducks exhibit a deletion of the "TTGAGTT" fragment, thus enabling accurate identification of Muscovy ducks using this Indel marker.

[0028] The nucleotide sequence containing the Indel label is preferably as shown in SEQ ID NO.1 or SEQ ID NO.2; the nucleotide sequence of SEQ ID NO.1 is:

[0029] The nucleotide sequence of SEQ ID NO.2 is as follows:

[0030] In SEQ ID NO.1 and SEQ ID NO.2, there are also differences in 8 SNP sites, indicated by bold and underlined letters. Specifically, SEQ ID NO.1 and SEQ ID NO.2 differ in one "TTGAGTT" fragment and 8 SNP sites. In the non-Muscovy duck sequence, there is an insertion of the "TTGAGTT" fragment and the corresponding bases of the 8 SNP sites are TGGCTATA. In the Muscovy duck sequence, there is not only a deletion of the "TTGAGTT" fragment, but the corresponding bases of the 8 SNP sites are AAAATAGCT. The "TTGAGTT" fragment alone can be used to accurately identify Muscovy ducks, and the differences in the 8 SNP sites can be used to assist in identification.

[0031] This invention provides primers for amplifying the Indel marker described in the above-mentioned technical solution. The primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, specifically 5'-GCAATAAAGGGGAATACC-3'. The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, specifically 5'-GACAAAGTGCGAAAGAGA-3'. The primers can specifically amplify the Indel marker, thereby achieving accurate identification of Muscovy duck species.

[0032] This invention provides a detection kit containing the primers and PCR amplification reagents described in the above-mentioned technical solution. The PCR amplification reagents of this invention preferably include DNA polymerase, dNTPs, and Mg. 2+ The DNA polymerase, dNTPs, and Mg 2+ There are no special requirements regarding the source or dosage; technologies well-known in the field can be used.

[0033] This invention provides the application of the Indel marker, primers, or detection kit described in the above technical solution in the screening and / or identification of Muscovy ducks. Experiments have shown that, by using the technical solution provided by this invention, Muscovy duck species can be specifically identified and / or screened.

[0034] This invention provides a method for screening and / or identifying Muscovy ducks, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain PCR amplification products; sequencing the PCR amplification products and making a judgment; when the sequence of the PCR amplification product contains the insertion of the "TTGAGTT" fragment, preferably in the duck RASEF gene at position 11096bp to 11102bp (in the second intron), the sample to be tested is determined not to be a Muscovy duck; when the sequence of the PCR amplification product does not contain the "TTGAGTT" fragment, preferably in the duck RASEF gene at position 11096bp to 11102bp (in the second intron), the sample to be tested is determined to be a Muscovy duck.

[0035] In this invention, when the PCR amplification product sequence contains the insertion of the "TTGAGTT" fragment, the sequence of the PCR amplification product is preferably as shown in SEQ ID NO.2; when the PCR amplification product sequence does not contain the "TTGAGTT" fragment, the sequence of the PCR amplification product is preferably as shown in SEQ ID NO.1; the source of the sample to be tested preferably includes one or more of blood, tissue, and cells; the tissue preferably includes embryonic tissue samples or skin tissue samples; there are no special requirements for the extraction method of the genomic DNA of the sample to be tested, and techniques well known in the art can be used.

[0036] The PCR amplification reaction system of the present invention, in 25 μL, preferably includes: 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L upstream primer, 0.5 μL of 10 μmol / L downstream primer, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O; the preferred PCR amplification reaction program is: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; and 72℃ extension for 5 min.

[0037] Experiments have shown that by using the technical solution provided by this invention, Muscovy ducks can be specifically screened from Beijing ducks, Aobaixing ducks, Cherry Valley ducks, Liancheng white ducks, Youxian Muscovy ducks, Jingjiang Muscovy ducks, Mountain Muscovy ducks, Shaoxing ducks, Jinyun Muscovy ducks, Mianyang Muscovy ducks, and Jinding ducks, with accurate, simple, and quick results.

[0038] To further illustrate the present invention, the application of an Indel marker, primer, and detection kit provided by the present invention in screening and / or identifying Muscovy ducks is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Preparation before the experiment:

[0040] Primer design: Based on the duck RASEF gene information (Gene ID: 101803543 on NCBI), primers containing partial sequences of the first intron, the complete sequence of the second exon, and partial sequences of the second intron were designed as follows:

[0041] Upstream primer RL (SEQ ID NO.3): 5'-GCAATAAAGGGGAATACC-3';

[0042] Downstream primer RR (SEQ ID NO.4): 5'-GACAAAGTGCGAAAGAGA-3'.

[0043] Example 1: Development of an Indel marker for Muscovy duck identification

[0044] 1. Samples to be tested: Blood DNA was extracted from 12 breeds of ducks preserved in the laboratory, including Beijing duck, Aobaixing duck, Cherry Valley duck, Liancheng white duck, Youxian Ma duck, Jingjiang Ma duck, Mountain Ma duck, Shaoxing duck, Jinyun Ma duck, Mianyang Ma duck, Jinding duck, and Muscovy duck. Blood DNA was extracted from these 12 breeds using the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd., and the concentration was determined.

[0045] 2. PCR amplification: For the above 12 varieties, 8 individuals were randomly selected from each variety for DNA samples (each individual's DNA concentration was 50 ng / μL). 10 μL of each of the 8 DNA samples from each variety was mixed to form a DNA sample, which served as the DNA template for that variety. In this way, DNA templates for 12 varieties were obtained. PCR amplification was performed using the upstream primer RL (SEQ ID NO.3) and the downstream primer RR (SEQ ID NO.4).

[0046] The PCR amplification reaction system, in 25 μL volume, consisted of 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L RL, 0.5 μL of 10 μmol / L RR, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O.

[0047] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0048] 3. Sequence analysis: The PCR products of the DNA templates of the above 12 varieties were detected by 1.2 wt.% agarose gel electrophoresis. The purified target bands were recovered from the agarose gel and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the peaks of all sequencing samples were clear and there were no impurity peaks.

[0049] In addition, the nucleotide sequences of the PCR products from DNA templates of 11 breeds, including Beijing Duck, Aobaixing Duck, Cherry Valley Duck, Liancheng White Duck, Youxian Ma Duck, Jingjiang Ma Duck, Mountain Ma Duck, Shaoxing Duck, Jinyun Ma Duck, Mianyang Ma Duck, and Jinding Duck, are all as shown in SEQ ID NO. 2, meaning that the sequencing results all contain a "TTGAGTT" sequence and 8 SNP sites. Figure 1 and Figure 2 Using the Peking duck as an example, the sequencing peak diagram of non-Muscovy ducks is shown (in...). Figure 2 In the image, the black box contains 8 SNPs (TGGCTATA), and the nucleotide sequence of the PCR product of the Muscovy duck DNA template is shown in SEQ ID NO.1. Figure 3 The sequence shown is a sequence that does not contain the sequence "TTGAGTT". Figure 4 The image shows eight SNPs, AAAATAGCT, from Muscovy duck sequencing results, which correspond to TGGCTATA in non-Muscovy duck sequences.

[0050] Example 2: Verification of the Indel marker for Muscovy ducks

[0051] 1. Blood samples were collected from five breeds of ducks: Cherry Valley Duck, Liancheng White Duck, Jingjiang Ma Duck, Mianyang Ma Duck, and Jinding Duck. Ten individuals from each breed were collected. DNA was extracted from the blood samples using the Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd., and the concentration was determined.

[0052] 2. PCR amplification: Using DNA from the above 50 individuals (10 DNA samples from each of 5 varieties) as templates, PCR amplification was performed using the upstream primer RL (SEQ ID NO.3) and the downstream primer RR (SEQ ID NO.4).

[0053] The PCR amplification reaction system consisted of 25 μL of 2×TaqPCRmix 12.5 μL, 10 μmol / L RL 0.5 μL, 10 μmol / L RR 0.5 μL, 50 ng / μL DNA template 1 μL, and ddH2O 10.5 μL.

[0054] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0055] 3. Sequence Analysis: PCR products from 50 individuals of the above 5 breeds were analyzed by 1.2 wt.% agarose gel electrophoresis. The purified target bands were recovered from the agarose gels and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the nucleotide sequences of the PCR products from all 50 individuals of the five breeds (Cherry Valley Duck, Liancheng White Duck, Jingjiang Ma Duck, Mianyang Ma Duck, and Jinding Duck) contained the sequence "TTGAGTT", and the 8 SNP sites were TGGCTATA. Figure 5 Using Cherry Valley ducks as an example, this demonstrates a sequence from a non-Muscovy duck sequencing peak diagram.

[0056] Example 3: Application of Indel markers in Muscovy duck identification

[0057] 1. 40 embryonic tissue samples of white-feathered Muscovy ducks: Embryonic tissue samples were collected from 40 individual white-feathered Muscovy ducks. DNA was extracted using the blood / cell / tissue genomic DNA extraction kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd., and the concentration was determined to obtain 40 DNA templates for testing.

[0058] 2. PCR amplification: Using the DNA from the above 40 Muscovy ducks as templates, PCR amplification was performed using the upstream primer RL (SEQ ID NO. 3) and the downstream primer RR (SEQ ID NO. 4).

[0059] The PCR amplification reaction system, in 25 μL volume, consisted of 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L RL, 0.5 μL of 10 μmol / L RR, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O.

[0060] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0061] 3. Sequence Analysis: PCR products from 40 Muscovy duck DNA templates were analyzed by 1.2 wt.% agarose gel electrophoresis. The purified target bands were recovered from the agarose gels and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown below. Figure 6 , Figure 6 This image shows a sequence from the sequencing peak diagram of a Muscovy duck.

[0062] Sequencing results showed that none of the 40 white-feathered Muscovy duck samples contained the "TTGAGTT" sequence, and the 8 SNP sites were: AAAATAGCT.

[0063] Example 4: Application of Indel markers in Muscovy duck identification

[0064] 1. Ten skin tissue samples from Muscovy ducks: Skin tissue samples were collected from 10 individual Muscovy ducks. DNA was extracted using the blood / cell / tissue genomic DNA extraction kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd., and the concentration was determined to obtain 10 DNA templates for testing.

[0065] 2. PCR amplification: Using the DNA from the 10 Muscovy ducks mentioned above as templates, PCR amplification was performed using the upstream primer RL (SEQ ID NO.3) and the downstream primer RR (SEQ ID NO.4).

[0066] The PCR amplification reaction system, in 25 μL volume, consisted of 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L RL, 0.5 μL of 10 μmol / L RR, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O.

[0067] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0068] 3. Sequence Analysis: The PCR products of 10 Muscovy duck DNA templates were analyzed by 1.2 wt.% agarose gel electrophoresis. The purified target bands were recovered from the agarose gels and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown below. Figure 7 , Figure 7 This image shows a sequence from the sequencing peak diagram of a Muscovy duck.

[0069] Sequencing results showed that none of the 10 Muscovy duck samples contained the "TTGAGTT" sequence, and the 8 SNP sites were AAAATAGCT.

[0070] Example 5: Application of Indel markers in Muscovy duck identification

[0071] 1. Twenty blood DNA samples from Yangxin Muscovy ducks: Blood samples were collected from 20 individual Yangxin Muscovy ducks. DNA was extracted using the blood / cell / tissue genomic DNA extraction kit (DP304) from Tiangen Biotech (Beijing) Co., Ltd., and the concentration was determined to obtain 20 DNA templates for testing.

[0072] 2. PCR amplification: Using the DNA from the above 20 Yangxin Muscovy ducks as templates, PCR amplification was performed using the upstream primer RL (SEQ ID NO.3) and the downstream primer RR (SEQ ID NO.4).

[0073] The PCR amplification reaction system, in 25 μL volume, consisted of 12.5 μL of 2×Taq PCR mix, 0.5 μL of 10 μmol / L RL, 0.5 μL of 10 μmol / L RR, 1 μL of 50 ng / μL DNA template, and 10.5 μL of ddH2O.

[0074] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 52℃ annealing for 20 s, 72℃ extension for 20 s, for 35 cycles; 72℃ extension for 5 min.

[0075] 3. Sequence Analysis: PCR products from 20 Yangxin Muscovy duck DNA templates were analyzed by 1.2 wt.% agarose gel electrophoresis. The purified target bands were recovered from the agarose gels and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown below. Figure 8 , Figure 8 This image shows a sequence from the sequencing peak diagram of Yangxin Muscovy ducks.

[0076] Sequencing results showed that none of the Muscovy duck samples contained the "TTGAGTT" sequence, and the eight SNP sites were: AAAATAGCT.

[0077] In summary, by adopting the technical solution provided by this invention, Muscovy ducks can be specifically screened and / or identified from Beijing ducks, Aobaixing ducks, Cherry Valley ducks, Liancheng white ducks, Youxian Muscovy ducks, Jingjiang Muscovy ducks, Mountain Muscovy ducks, Shaoxing ducks, Jinyun Muscovy ducks, Mianyang Muscovy ducks, and Jinding ducks, with accurate, simple, and fast results.

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method of screening and / or identifying Call Ducks, characterized in that, The method comprises the following steps: Taking genomic DNA of the sample to be tested as a template, performing PCR amplification by using an upstream primer and a downstream primer to obtain a PCR amplification product; performing sequencing on the PCR amplification product and making a judgment; When the sequence of the PCR amplification product is as shown in SEQ ID NO. 2, it is determined that the sample to be tested is not Muscovy duck; When the sequence of the PCR amplification product is as shown in SEQ ID NO. 1, it is determined that the sample to be tested is Muscovy duck; The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.

3. The nucleotide sequence of the downstream primer is as shown in SEQ ID NO.

4.

2. The method of claim 1, wherein, The sample to be tested is derived from blood and / or tissue.

3. The method according to claim 1 or 2, characterized in that, The reaction system of the PCR amplification, in the amount of 25 μL, comprises the following: 2×Taq PCR mix 12.5 μL, 10 μmol / L upstream primer 0.5 μL, 10 μmol / L downstream primer 0.5 μL, 50 ng / μL DNA template 1 μL, and ddH2O 10.5 μL.

4. The method according to claim 1 or 2, characterized in that, The reaction procedure of the PCR amplification is as follows: 98 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 20 s, 72 ℃ extension for 20 s, 35 cycles; and 72 ℃ extension for 5 min.

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