Primer composition for detecting acipenser Yangtze River MNP marker sites, kit and application of primer composition and kit

Abstract

The invention provides a primer composition and a kit for detecting acipenser Yangtze River MNP marker sites and application of the primer composition and the kit. Wherein the primer composition comprises 100 pairs of primer pairs, and the nucleotide sequences of the 100 pairs of primer pairs are as shown in SEQ ID NO: 1 to SEQ ID NO: 200. The primer composition not only can be used for identifying the species or individuals of the Yangtze River sturgeons, but also can be used for identifying the species or genetic diversity of the Yangtze River sturgeons in a water area based on the DNA of a water environment, has the advantages of high flux, strong distinction degree, good accuracy and high accuracy, and provides technical support for scientific research, fishery monitoring and ecological system investigation of the Yangtze River sturgeons.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer composition, a kit and applications thereof for detecting a Yangtze sturgeon MNP marker site. Background Art

[0002] The Yangtze sturgeon (Acipenser dabryanus) belongs to the order Acipenseriformes, family Acipenseridae, genus Acipenser. It is mainly distributed in the main and tributary rivers of the Yangtze River. It is a large freshwater sedentary fish unique to China. Due to the impact of human activities, the wild resources of the Yangtze sturgeon have declined sharply. It was listed in the Convention on International Trade in Endangered Species in 2020. It is urgent to conduct a survey and restoration of the wild resources of the Yangtze sturgeon. Compared with traditional fishing methods, the operation of environmental DNA technology is relatively simple, and the way of collecting water samples is more conducive to large-scale or multi-frequency monitoring, which will not cause direct interference or harm to fish. Therefore, it has been widely used in fish monitoring surveys in recent years.

[0003] The application of environmental DNA technology in fish mainly includes two aspects: one is the investigation of a single fish species, and the other is the investigation of fish diversity. The current environmental DNA technology is based on mitochondrial DNA. For example, COI, 16S, 12S or CytB fragments are used to design specific primers for a single fish species or universal primers that can be used for fish diversity analysis, and then combined with qPCR, ddPCR or high-throughput sequencing to complete a single fish species investigation or a new analysis of fish diversity. Since DNA is easily degraded in natural water bodies, the target fragments of the above-mentioned single primer markers such as COI, 16S, 12S, and CytB are generally set at 100-300bp. The shorter target fragments make it difficult for this technology to distinguish closely related fish species; in addition, some rare, endangered or naturally small fish species, due to the low concentration of DNA released in the water, lead to the filtering of sequence information during environmental DNA monitoring, and often false negative problems. In short, the low content of DNA in water bodies and easy fragmentation may cause problems with insufficient monitoring accuracy and sensitivity. In order to give full play to the technical advantages of environmental DNA, it is necessary to develop new highly polymorphic molecular markers and their detection technologies based on water environment samples. Summary of the invention

[0004] The purpose of the present invention is to provide a primer composition, a kit and its application for detecting the MNP marker site of Yangtze sturgeon, and at the same time, to propose a method for identifying Yangtze sturgeon species based on water environment DNA. The above invention content can not only meet the needs of Yangtze sturgeon species identification and individual identification, but also achieve the purpose of Yangtze sturgeon species identification and genetic diversity analysis based on water environment DNA, and has the advantages of high throughput, strong discrimination, good precision and high accuracy.

[0005] The present invention provides a primer composition for detecting Yangtze sturgeon MNP marker sites, which comprises 100 pairs of primer pairs, and the nucleotide sequences of the 100 pairs of primer pairs are shown in SEQ ID NO: 1-SEQ ID NO: 200.

[0006] The present invention provides a kit for detecting the MNP marker site of Yangtze sturgeon, which comprises the above-mentioned primer combination.

[0007] The kit as described above, wherein the kit further comprises at least one of water, multiplex PCR buffer, dNTPs, and DNA polymerase.

[0008] The present invention provides an application of the primer combination or the kit in the identification of Yangtze sturgeon species.

[0009] The present invention provides an application of the primer combination or the kit in the identification of Yangtze sturgeon individuals.

[0010] The present invention provides an application of the primer combination or the kit in Yangtze River sturgeon species identification based on water environment DNA.

[0011] The present invention provides an application of the primer combination or the kit in genetic diversity analysis of Yangtze sturgeon based on water environment DNA.

[0012] The present invention provides a method for identifying Yangtze sturgeon species based on water environment DNA, which comprises the following steps:

[0013] Obtaining a water environment sample from a water area to be tested, extracting DNA from the water environment sample, and obtaining water environment DNA;

[0014] Using the water environment DNA as a template, using the above primer combination or the above kit to perform multiplex PCR amplification to obtain multiplex PCR amplification products;

[0015] The multiplex PCR amplification products were sequenced with high throughput to obtain the primer-labeled sites of the water environment DNA, and the Yangtze sturgeon species in the waters to be tested were identified based on the primer-labeled sites.

[0016] The method as described above, wherein the multiplex PCR amplification includes a first round of multiplex PCR amplification and a second round of multiplex PCR amplification;

[0017] The reaction system for the first round of multiplex PCR amplification includes: 5 μL of water environment DNA, 4 μL of the above primer combination or the above kit, 10 μL of GenoPlexs 3×T Master Mix and 11 μL of ddH 2 O;

[0018] The reaction system for the second round of multiplex PCR amplification included: 16 μL of the first round of multiplex PCR amplification product, 4 μL of 5 μM Illumina sequencing adapter, and 10 μL of GenoPlexs 3×T Master Mix.

[0019] The method as described above, wherein the reaction procedure of the first round of multiplex PCR amplification includes: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, for a total of 18 cycles; extension at 72°C for 4 min;

[0020] The reaction procedure of the second round of multiplex PCR amplification included: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 70°C for 30 s, for a total of 8 cycles; and final extension at 72°C for 5 min.

[0021] The present invention provides a primer composition, a kit and an application thereof for detecting a Yangtze sturgeon MNP marker site, wherein the primer composition comprises 100 pairs of primer pairs, and the nucleotide sequences of the 100 pairs of primer pairs are shown in SEQ ID NO: 1-SEQ ID NO: 200. The primer composition can be used to identify not only Yangtze sturgeon species or individuals, but also Yangtze sturgeon species or genetic diversity in water areas based on water environment DNA, with the advantages of high throughput, strong discrimination, good precision and high accuracy, and provides technical support for scientific research, fishery monitoring and ecosystem investigation of Yangtze sturgeon. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The genetic cluster analysis diagram of Yangtze sturgeon and other sturgeons in Example 3;

[0023] Figure 2 This is a distribution diagram of the difference ratio of MNP marker sites between Yangtze sturgeon individuals in Example 4. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0025] There are multiple SNP sites in the genome, and the multiple nucleotide polymorphism (MNP) marker technology can combine the alleles of these different SNP sites to distinguish different individual DNA. MNP marker technology is a newly emerging method for plant variety identification after the simple sequence repeat (SSR) marker technology and single nucleotide polymorphism (SNP) marker technology. It has achieved significant results in plant variety identification and has been successfully applied to the variety identification of crops such as rice, soybean, rapeseed, eggplant, corn, and tomato. At present, there are few studies on the application of MNP marker technology in fish identification. The fish genome is relatively complex, with a large number of repeated sequences and hybridization phenomena, and it is also difficult to obtain a sufficient number of rare and endemic fish samples. Therefore, it is difficult to develop high-quality molecular marker technology for fish diversity identification.

[0026] To solve the above problems, the first aspect of the present invention provides a primer composition for detecting MNP marker sites of Yangtze sturgeon, comprising 100 pairs of primer pairs, and the nucleotide sequences of the 100 pairs of primer pairs are shown in SEQ ID NO: 1-SEQ ID NO: 200.

[0027] The present invention aims at the difficulty of environmental DNA (eDNA) in fish identification, and develops a primer combination for detecting the MNP marker site of Yangtze sturgeon. The primer combination can specifically amplify the MNP marker site of Yangtze sturgeon, and the primers do not interfere with each other, and can be efficiently amplified in a single reaction pool. It has the advantages of high flux, strong discrimination, good precision, and high accuracy. It can not only identify the species or individuals of Yangtze sturgeon, but also identify the species or genetic diversity of Yangtze sturgeon in water areas based on water environment DNA. The present invention provides technical support for research such as individual tracking of Yangtze sturgeon and evaluation of release effects, helps to comprehensively evaluate the ecological status of Yangtze sturgeon in the entire aquatic ecosystem, solves the industry problem that environmental DNA technology is difficult to distinguish closely related fish species, broadens the application scope of environmental DNA, and also provides research data for the application of MNP marker technology in animal identification.

[0028] The second aspect of the present invention provides a kit for detecting the MNP marker site of Yangtze sturgeon, comprising the above-mentioned primer combination.

[0029] Since the kit includes the primer combination of the present invention, the kit can also specifically amplify the Yangtze sturgeon MNP marker site, and has the advantages of high throughput, strong discrimination, good precision and high accuracy.

[0030] In the above technical solution, the kit further includes at least one of water, multiple PCR buffer, dNTPs, and DNA polymerase.

[0031] Among them, water can provide the ion environment and water molecule medium required for the multiplex PCR reaction, so that the interaction between various molecules in the reaction can proceed smoothly, and it can also help maintain the temperature stability of the reaction system to ensure the reliability and accuracy of the reaction. The multiplex PCR buffer can adjust the pH value of the reaction system to keep the working environment of the DNA polymerase alkaline. 2+ It can also affect the specificity of the reaction and the yield of the amplified fragment. dNTPs refers to four free deoxyribonucleoside triphosphates, which are the raw materials necessary for PCR amplification. DNA polymerase refers to a class of enzymes that use parent DNA as a template to catalyze the polymerization of substrate dNTPs molecules to form daughter DNA.

[0032] A third aspect of the present invention provides a use of the above-mentioned primer combination or the above-mentioned kit in the identification of Yangtze sturgeon species.

[0033] In a specific embodiment, the identification of Yangtze sturgeon species first requires obtaining the genomic DNA of the sample to be detected, and then using the above primer combination or the above kit to perform a first round of multiple PCR amplification (cycle number not higher than 25) on the genomic DNA, followed by purification of the amplified product of the multiple PCR, and then adding a sequencing adapter thereto and performing a second round of multiple PCR amplification (cycle number not higher than 10), to obtain a high-throughput sequencing library of the sample to be detected, purify and sequence the high-throughput sequencing library, and obtain sequencing data. The sequencing data is compared with the reference sequence of the Yangtze sturgeon to obtain the sequence of the sample to be detected at the above Yangtze sturgeon MNP marker site, thereby analyzing the number of detection sites and genotype data of the sample to be detected at the MNP site. Finally, genetic cluster analysis of the number of detection sites and genotype data of the sample to be detected at the MNP site can determine whether the sample to be detected is the Yangtze sturgeon or other sturgeons (such as small-bodied sturgeon, Chinese sturgeon).

[0034] A fourth aspect of the present invention provides a use of the above-mentioned primer combination or the above-mentioned kit in the identification of Yangtze sturgeon individuals.

[0035] In a specific embodiment, the identification of Yangtze sturgeon individuals requires obtaining the number of detection sites and genotype data of the sample to be tested at the MNP site, comparing the above data with the number of detection sites and genotype data of the candidate Yangtze sturgeon sample at the MNP site and calculating the genetic similarity. When the genetic similarity between the sample to be tested and the candidate Yangtze sturgeon sample is ≥99%, the sample to be tested and the candidate Yangtze sturgeon sample are derived from the same Yangtze sturgeon individual. The primer combination or kit provided by the present invention has a strong ability to distinguish individuals of Yangtze sturgeon and is very suitable for the identification of Yangtze sturgeon individuals.

[0036] A fifth aspect of the present invention provides an application of the above-mentioned primer combination or the above-mentioned kit in the identification of Yangtze sturgeon species based on water environment DNA.

[0037] Among them, water environment DNA refers to a mixed DNA sample isolated from water environment samples collected from the water area to be tested. Its sources include biological relics such as epidermal cells, excrement, mucus and body surface appendages shed by organisms in the water area, which are released into the water environment through biological metabolism or physical diffusion.

[0038] Therefore, the identification of Yangtze sturgeon species based on water environment DNA has the advantages of being simple and quick to operate, and the way of collecting water samples is more conducive to large-scale or multi-frequency monitoring, and will not cause direct interference or harm to fish. It can be widely used in fish monitoring surveys and has good application prospects. After experimental verification, the primer combination or kit developed by the present invention can be used for the identification of Yangtze sturgeon species based on water environment DNA, and can effectively determine the Yangtze sturgeon species in the waters to be tested, with the advantages of high flux, strong discrimination, good precision and high accuracy. The sixth aspect of the present invention provides an application of the above-mentioned primer combination or the above-mentioned kit in the genetic diversity analysis of Yangtze sturgeon based on water environment DNA.

[0039] In a specific embodiment, the genetic diversity analysis of Yangtze sturgeon based on water environment DNA requires obtaining the sequence of the water environment sample at the above-mentioned Yangtze sturgeon MNP marker site, and obtaining the average allele number by analyzing and calculating the above-mentioned sequence. The high or low average allele number in the water environment sample can reflect the high or low genetic diversity of Yangtze sturgeon in the corresponding water area.

[0040] A seventh aspect of the present invention provides a method for identifying Yangtze sturgeon species based on water environment DNA, comprising the following steps:

[0041] Obtaining a water environment sample from a water area to be tested, extracting DNA from the water environment sample, and obtaining water environment DNA;

[0042] Using the water environment DNA as a template, using the above primer combination or the above kit to perform multiplex PCR amplification to obtain multiplex PCR amplification products;

[0043] The multiplex PCR amplification products were sequenced with high throughput to obtain the primer-labeled sites of the water environment DNA, and the Yangtze sturgeon species in the waters to be tested were identified based on the primer-labeled sites.

[0044] First, the present invention does not limit how to extract DNA from water environment samples, and those skilled in the art can perform extraction according to conventional methods. In a specific embodiment, a universal DNA extraction kit for environmental samples with a product number of TD430-50 produced by Jianshi Biological Company can be used for extraction.

[0045] Secondly, using the water environment DNA as a template, multiplex PCR amplification is performed using the above primer combination or the above kit, with the number of cycles being no higher than 25, the main purpose of which is to enrich the target MNP marker.

[0046] Specifically, the multiplex PCR amplification includes a first round of multiplex PCR amplification and a second round of multiplex PCR amplification. The reaction system of the first round of multiplex PCR amplification includes: 5 μL of water environment DNA, 4 μL of the above primer combination or the above kit, 10 μL of GenoPlexs3×T Master Mix and 11 μL of ddH 2 O. Among them, 4 μL of the above-mentioned primer composition or the above-mentioned kit can be prepared by the following method: 5 μL (0.2 μM) of each primer in 100 pairs of primers (i.e., 200 primers) with nucleotide sequences as shown in SEQ ID NO: 1-SEQ ID NO: 200 is mixed to prepare 1 mL of primer premix, and then 4 μL of the above-mentioned primer composition or the above-mentioned kit is added to the reaction system of the first round of multiple PCR amplification.

[0047] In the above technical solution, the reaction procedure of the first round of multiplex PCR amplification includes: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 4 minutes, a total of 18 cycles; and extension at 72°C for 4 minutes.

[0048] In order to perform high-throughput sequencing, the products of the first-round multiplex PCR amplification need to be purified, and then the sequencing adapters are connected to the MNP marker sequences enriched in the first-round multiplex PCR through a second-round PCR amplification, with the number of cycles being no higher than 10. The obtained amplified products can be used as high-throughput sequencing libraries.

[0049] Specifically, the reaction system of the second round of multiplex PCR amplification includes: 16 μL of purified multiplex PCR amplification products, 4 μL of Illumina sequencing adapters with a concentration of 5 μM, and 10 μL of GenoPlexs 3×T Master Mix. The reaction procedure of the second round of multiplex PCR amplification includes: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 70°C extension for 30 s, a total of 8 cycles; 72°C final extension for 5 min.

[0050] Finally, in the present invention, the multiplex PCR amplification products can be subjected to high-throughput sequencing to obtain the primer-labeled sites of the water environment DNA, and the Yangtze sturgeon species in the water area to be detected can be identified according to the primer-labeled sites.

[0051] Specifically, the primer marker site of the water environment DNA can be compared with the reference sequence of the Yangtze sturgeon (the Yangtze sturgeon MNP marker site), and the sequence of the water environment sample at the Yangtze sturgeon MNP marker site can be obtained, so as to analyze and obtain the number of detection sites of the water environment sample at the MNP site. Finally, according to the number of detection sites of the water environment sample at the MNP site, it can be analyzed whether the Yangtze sturgeon species exists in the water area to be tested.

[0052] The technical solution of the present application will be further explained below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The reagents used are commercially available or available from public channels unless otherwise specified.

[0053] Example 1: Design of multiplex PCR primer composition

[0054] DNA was extracted from 30 Yangtze sturgeons from the Chinese Sturgeon Research Institute of China Three Gorges Corporation to obtain samples to be tested. Simplified genome sequencing was then performed on the samples to be tested to obtain sequencing data for each sample. Samtools (version 1.2) and BCFtools (version 1.2) sequence analysis software were used to perform sequence analysis on the sequencing data of the samples to be tested, and the sequencing data of the samples to be tested were compared with the reference gene sequence (SRR15851056) to obtain the SNP sites of the Yangtze sturgeon, and the comparison results were saved in SAM (The Sequence Alignment / Map format). The SNP site was compared with the Nucleotide Sequence Database (NT) of the National Center of Biotechnology Information (NCBI) of the United States, and the Yangtze sturgeon MNP marker site was selected according to the following principles: (1) The sequence of the Yangtze sturgeon MNP marker site is unique to the Yangtze sturgeon and does not appear in other species; (2) The sequence of the Yangtze sturgeon MNP marker site is a single copy in the genome; (3) There are at least three discontinuous SNP differences in the sequence of the Yangtze sturgeon MNP marker site; (4) The sequence length of the Yangtze sturgeon MNP marker site is 100-300bp. After the candidate Yangtze sturgeon MNP marker site was screened, the discrimination of the candidate Yangtze sturgeon MNP marker site was analyzed using the sequencing data of the above-mentioned sample to be tested, and finally 100 Yangtze sturgeon MNP marker sites were obtained.

[0055] According to the sequences of the 100 Yangtze sturgeon MNP marker sites obtained, a multiple polymerase chain reaction (PCR) primer pair corresponding to each Yangtze sturgeon MNP marker site was designed, and the primer pair included an upstream primer and a downstream primer; that is, the multiple PCR primer composition designed according to the 100 Yangtze sturgeon MNP marker sites includes 100 pairs of primer pairs, containing a total of 200 primers. Among them, the design of primers needs to follow the principle of non-interference between primers, and all primers can be amplified normally in one amplification reaction. The upstream primer and downstream primer sequences for each Yangtze sturgeon MNP marker site can be seen in Table 1.

[0056] Table 1

[0057]

[0058]

[0059]

[0060]

[0061] Example 2: Evaluation of Yangtze sturgeon MNP marker loci and multiplex PCR primer combinations

[0062] In this example, 30 Yangtze sturgeons in the above-mentioned Example 1 were selected as evaluation samples to evaluate the Yangtze sturgeon MNP marker sites and multiplex PCR primer combinations. The specific method is as follows:

[0063] (1) DNA extraction: The tissue DNA of the above-mentioned evaluation samples was extracted using the animal tissue genomic DNA extraction kit with the catalog number DP324 produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain DNA samples. 1 μL of the DNA sample was taken from each sample using a Qubit fluorescence quantifier to determine the DNA concentration, ensuring that the DNA concentration in the DNA sample was 20-40 ng / μL.

[0064] (2) Multiplex PCR: Take 5 μL (0.2 μM) of each primer in the multiplex PCR primer composition obtained in Example 1 and mix them evenly to prepare 1 mL of primer composition solution for use. Use the primer composition solution to perform multiplex PCR amplification on the DNA sample obtained in step (1) to obtain multiplex PCR amplification products. The multiplex PCR reaction system includes 5 μL of DNA sample, 4 μL of primer composition solution, 10 μL of GenoPlexs 3×T MasterMix produced by Shijiazhuang Boridi Biotechnology Co., Ltd., and 11 μL of ddH 2O. The multiplex PCR amplification procedure included 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 60°C annealing for 4 min, for a total of 18 cycles; and finally 72°C extension for 4 min. After the reaction, the multiplex PCR amplification products were stored at 4°C.

[0065] (3) Purification of multiplex PCR products: The multiplex PCR amplification products obtained in step (2) were purified using magnetic beads with product number N411 produced by Nanjing Novozymes Biotech Co., Ltd. to obtain purified multiplex PCR amplification products.

[0066] (4) Construction of high-throughput sequencing library: The sequencing adapters were connected to the purified multiplex PCR amplification products obtained in step (3) by PCR amplification to obtain a high-throughput sequencing library. The PCR reaction system included 16 μL of purified multiplex PCR amplification products, 10 μL of GenoPlexs 3×T Master Mix, and 4 μL of Illumina sequencing adapters with a concentration of 5 μM. The PCR amplification procedure included pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 70°C for 30 s, for a total of 8 cycles; and final extension at 72°C for 5 min.

[0067] (5) Purification of high-throughput sequencing library: The high-throughput sequencing library obtained in step (4) was purified using magnetic beads with catalog number N411 produced by Nanjing Novozymes Biotech Co., Ltd. to obtain a purified high-throughput sequencing library.

[0068] (6) Sequencing: The purified high-throughput sequencing library obtained in step (5) is sequenced using an Illumina NextSeq1000 sequencer. The detailed sequencing steps are performed according to the instruction manual of the sequencer. Sequencing data is obtained after sequencing is completed.

[0069] (7) Sequencing data analysis: The sequencing data obtained in step (6) was aligned to the reference sequence of Example 1 using the data alignment software Bowtie2 (version 2.1.0). The alignment results were saved in the SAM (Sequence Alignment / Map format) format to obtain the primer marker sequence at the Yangtze sturgeon MNP marker site of the evaluation sample.

[0070] (8) Detection rate of Yangtze sturgeon MNP marker sites: The primer marker sequences at 100 Yangtze sturgeon MNP marker sites of 30 evaluation samples were compared with the reference sequence, and a total of 3000 Yangtze sturgeon MNP marker sites were compared. When the match rate between the sequence of the evaluation sample at the Yangtze sturgeon MNP marker site and the reference sequence met the default requirement of the software Bowtie2 (≥90%), the Yangtze sturgeon MNP marker site was determined to be detected in the evaluation sample.

[0071] The number of detected MNP marker sites of 100 Yangtze sturgeons in 30 evaluation samples was counted, among which 100 Yangtze sturgeon MNP marker sites could be detected in each evaluation sample, with a detection rate of 100%. The above experimental results show that the detection method based on MNP site markers is highly efficient and can achieve the detection of 3,000 marker sites in one experiment, and it has a 100% detection rate for Yangtze sturgeon samples.

[0072] (9) Reproducibility and accuracy of multiplex PCR primer composition: 10 evaluation samples were randomly selected, and two DNA samples were extracted from each evaluation sample. Library 1 and Library 2 were constructed according to the method of steps (2)-(5), and Library 1 and Library 2 were sequenced and aligned. The alignment results of Library 1 and Library 2 were compared to detect whether the genotypes of Library 1 and Library 2 at the same Yangtze sturgeon MNP marker site were consistent. If the genotypes were consistent, it was proved that the Yangtze sturgeon MNP marker site had good reproducibility. At the same time, the typing precision and typing accuracy were calculated. Among them, the calculation formula of typing precision (r) is as follows:

[0073] r=M 1 / M 2 ×100%, where M 1 M is the number of marker sites detected by both library 1 and library 2 with no genotype difference. 2 The number of marker sites detected by both library 1 and library 2;

[0074] The calculation formula of typing accuracy (a) is as follows:

[0075] Where r is the typing accuracy.

[0076] Table 2

[0077]

[0078] As shown in Table 2, the detection method based on MNP site labeling was reproducible in both library construction and sequencing, with excellent precision and accuracy, with a reproducibility of 100% and an accuracy of 100%.

[0079] Example 3: Yangtze sturgeon species identification

[0080] The sturgeons provided by the Chinese Acipenser Research Institute of China Three Gorges Corporation were used as samples, among which the small-bodied sturgeon was numbered MY-1, the Chinese sturgeon was numbered MY-2, and the Yangtze sturgeon were numbered MY-3, MY-4, MY-5, MY-6, MY-7, and MY-8, respectively. The above sturgeon samples were tested according to steps (1)-(7) of Example 2 to obtain the primer marker sequences of the sturgeon samples at the Yangtze sturgeon MNP marker site. Genetic cluster analysis was performed based on the sequences of the sturgeon samples at the Yangtze sturgeon MNP marker site. The specific results can be seen Figure 1 , Figure 1 The height indicates the genetic distance.

[0081] like Figure 1 As shown, the Yangtze sturgeon, the small-bodied sturgeon and the Chinese sturgeon are clustered into different categories, and the six samples of the Yangtze sturgeon are significantly clustered into 1 category. The above results show that the 100 Yangtze sturgeon MNP marker sites provided by the present invention can effectively distinguish different sturgeons, and thus be used for the identification of Yangtze sturgeon species.

[0082] Example 4: Individual identification of Yangtze sturgeon

[0083] Randomly select one sample from MY-3, MY-4, MY-5, MY-6, MY-7, and MY-8 in Example 3 as the sample to be tested, named MY-9, and use MY-3, MY-4, MY-5, MY-6, MY-7, and MY-8 as the control group. MY-9 was tested according to steps (1) to (7) of Example 2 to obtain the primer marker sequence of MY-9 at the MNP marker site of the Yangtze sturgeon. The primer marker sequence of MY-9 at the MNP marker site of the Yangtze sturgeon was compared with the above-mentioned control group, and the number of sites with different sequences at the MNP marker site was counted, and the genetic similarity between the samples was calculated to obtain the identification conclusion. The specific results can be seen in Table 3, where the calculation formula of genetic similarity is as follows: V 1 / V 2 ×100%,V 1 V is the number of marker sites detected in both the test sample and the control group with no genotype difference. 2 It is the number of marker sites detected jointly in the test sample and the control group.

[0084] Table 3

[0085] Samples to be tested Control group Number of common detection sites Number of differential sites Genetic similarity (%) Identification conclusion MY-9 MY-3 100 53 47 Different individuals MY-9 MY-4 100 49 51 Different individuals MY-9 MY-5 100 0 100 Same individual MY-9 MY-6 100 45 55 Different individuals MY-9 MY-7 100 48 42 Different individuals MY-9 MY-8 100 47 53 Different individuals

[0086] As shown in Table 3, the genetic similarity between MY-9 and MY-5 is 100%, and they are identified as the same individual, which is consistent with the fact that the MY-9 and MY-5 samples are derived from the same Yangtze sturgeon; while the genetic differences between MY-9 and other samples are significantly less than 99%, and they are identified as different individuals. The above results show that the 100 Yangtze sturgeon MNP marker sites provided by the present invention can effectively distinguish different Yangtze sturgeon individuals, and thus be used for the identification of Yangtze sturgeon individuals.

[0087] In addition, the sequencing data of the six samples in the control group were compared one by one, that is, MY-3 was compared with MY-4, MY-5, MY-6, MY-7, and MY-8, MY-4 was compared with MY-5, MY-6, MY-7, and MY-8, MY-5 was compared with MY-6, MY-7, and MY-8, MY-6 was compared with MY-7 and MY-8, and MY-7 was compared with MY-8. A total of 15 sample comparison results were obtained, which can be seen in detail. Figure 2 .

[0088] Figure 2 The average difference ratio of the MNP marker sites in the 15 samples was 48.73%, indicating that the 100 Yangtze sturgeon MNP marker sites provided by the present invention have strong individual discrimination ability and are particularly suitable for the identification of Yangtze sturgeon individuals.

[0089] Example 5: Yangtze River sturgeon species identification based on water environment DNA

[0090] A Yangtze sturgeon tissue sample (MY10) provided by the Chinese Sturgeon Research Institute of China Three Gorges Corporation was set as a positive control group, and two water environment samples (T1-M1 and T1-M2) from the Yangtze sturgeon infestation area of ​​Yibin City provided by the Chinese Sturgeon Research Institute of China Three Gorges Corporation were set as experimental groups. DNA was extracted from the filtered experimental group using a universal DNA extraction kit for environmental samples with a product number of TD430-50 produced by Jianshi Biological Company, and tested according to the detection method of steps (2)-(7) of Example 2, and the positive control group was tested according to the detection method of steps (1)-(8) of Example 2. The test results can be seen in Table 4.

[0091] Table 4

[0092] Sample No. Sample Information Number of MNP marker sites detected MNP labeling site detection ratio (%) MY10 Yangtze sturgeon tissue samples 100 100 T1-M1 Water environment sample of the Yangtze River sturgeon infestation area in Yibin City 1 61 61 T1-M2 Water environment samples from the Yangtze River sturgeon infestation area in Yibin City 2 47 47

[0093] As shown in Table 4, 100 MNP marker sites of Yangtze sturgeon were detected in the positive control group, and 61 and 47 MNP marker sites of Yangtze sturgeon were detected in the experimental group, respectively, that is, Yangtze sturgeon species existed in the waters corresponding to water environment sample 1 in the area where Yangtze sturgeon infested in Yibin City, and in the waters corresponding to water environment sample 2 in the area where Yangtze sturgeon infested in Yibin City. This experimental result is consistent with the actual situation, indicating that the detection method based on MNP site markers can be used to identify Yangtze sturgeon species using water environment DNA as samples.

[0094] Example 6: Analysis of Yangtze River sturgeon diversity based on water environment DNA

[0095] The water environment sample obtained by sampling from the downstream of Xiangjia Dam, Jinsha River, Yibin City was named BX-R2, the water environment sample obtained by sampling from the mouth of Chishui River, Hejiang County was named HJ2-R3, and the water environment sample obtained by sampling from the downstream of Luzhou was named T1-L2. BX-R2, HJ2-R3, and T1-L2 were filtered to obtain samples to be tested, and DNA was extracted using a universal DNA extraction kit for environmental samples with a product number of TD430-50 produced by Jianshi Biological Company. Then, the test was performed according to the test method of steps (2)-(8) of Example 2. The test results can be seen in Table 5, where the calculation formula for the average allele number (Gd) of MNP markers is as follows:

[0096] Among them, A i is the allele number of the ith detected Yangtze sturgeon MNP marker site in the sample to be tested, and N is the number of Yangtze sturgeon MNP marker sites detected in the sample to be tested.

[0097] Table 5

[0098]

[0099] Since the higher the genetic diversity of fish, the more alleles it has. Therefore, by detecting the average number of alleles in water environment samples, the genetic diversity of Yangtze sturgeon in the corresponding waters can be reflected. As shown in Table 5, the average number of alleles of the MNP marker site of BX-R2 is higher than that of other samples, indicating that the genetic diversity of Yangtze sturgeon in the area downstream of Xiangjiaba, Jinsha River, Yibin City is higher than that in other areas. The above results show that the detection method based on MNP site markers can use water environment DNA as a sample to analyze the number and genetic diversity of Yangtze sturgeon in waters.

[0100] In summary, the present invention provides a Yangtze sturgeon MNP marker site, primer composition and kit and their applications. Among them, the MNP marker site is developed based on the Yangtze sturgeon reference sequence, and the high-throughput sequencing data of the Yangtze sturgeon is downloaded from NCBI to dig out the MNP site that is different from other species, and then the MNP site detection primers suitable for multiple PCR amplification are designed through the conservative sequences on both sides of the MNP site, and then a set of MNP sites with high polymorphism and strong specificity and the most compatible primer composition and kit are screened according to the test results of the standard. The present invention uses multiple PCR amplification technology and second-generation sequencing technology through primer compositions and kits to identify the MNP marker sites of the Yangtze sturgeon, which has the advantages of high throughput, high discrimination, good precision and high accuracy, can meet the needs of Yangtze sturgeon species identification and individual identification, and can also achieve the purpose of Yangtze sturgeon species identification and genetic diversity analysis based on water environment DNA, providing technical support for scientific research, fishery monitoring, and ecosystem investigation of the Yangtze sturgeon.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting a Yangtze sturgeon MNP marker site, characterized in that: It comprises 100 pairs of primers, and the nucleotide sequences of the 100 pairs of primers are shown as SEQ ID NO: 1-SEQ ID NO:

200.

2. A kit for detecting the MNP marker sites of Yangtze sturgeon, characterized in that: The invention comprises the primer composition as claimed in claim 1.

3. The kit according to claim 2, characterized in that The kit further comprises at least one of water, multiplex PCR buffer, dNTPs, and DNA polymerase.

4. Use of the primer composition according to claim 1 or the kit according to claim 2 or 3 in the identification of Yangtze sturgeon species.

5. Use of the primer composition according to claim 1 or the kit according to claim 2 or 3 in the identification of Yangtze sturgeon individuals.

6. Use of the primer composition described in claim 1 or the kit described in claim 2 or 3 in the identification of Yangtze sturgeon species based on water environmental DNA.

7. Use of the primer composition according to claim 1 or the kit according to claim 2 or 3 in genetic diversity analysis of Yangtze sturgeon based on water environment DNA.

8. A method for identifying Yangtze sturgeon species based on water environment DNA, characterized in that: The steps include: Obtaining a water environment sample from a water area to be tested, extracting DNA from the water environment sample, and obtaining water environment DNA; Using water environment DNA as a template, using the primer composition of claim 1 or the kit of claim 2 or 3 to perform multiplex PCR amplification to obtain multiplex PCR amplification products; The multiplex PCR amplification products are subjected to high-throughput sequencing to obtain primer-labeled sites of water environment DNA, and the Yangtze sturgeon species in the waters to be detected are identified based on the primer-labeled sites.

9. The method according to claim 8, characterized in that The multiplex PCR amplification includes a first round of multiplex PCR amplification and a second round of multiplex PCR amplification; The reaction system of the first round of multiplex PCR amplification includes: 5 μL of the water environment DNA, 4 μL of the primer composition of claim 1 or the kit of claim 2 or 3, 10 μL of GenoPlexs 3×T Master Mix and 11 μL of ddH2O; The reaction system for the second round of multiplex PCR amplification included: 16 μL of the first round of multiplex PCR amplification product, 4 μL of 5 μM Illumina sequencing adapter, and 10 μL of GenoPlexs 3×T Master Mix.

10. The method according to claim 9, characterized in that The reaction procedure of the first round of multiplex PCR amplification includes: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 4 min, for a total of 18 cycles; extension at 72°C for 4 min; The reaction procedure of the second round of multiplex PCR amplification includes: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 70°C for 30 s, for a total of 8 cycles; and final extension at 72°C for 5 min.

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