Primer composition, kit for detecting mnp marker site of acipenser dabryanus and application thereof

Abstract

The application provides a primer composition, a kit for detecting MNP marker sites of Yangtze sturgeon and application thereof. 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. By using the primer composition, not only the Yangtze sturgeon species or individual can be identified, but also the Yangtze sturgeon species or genetic diversity in the water area can be identified based on the water environment DNA, and the primer composition has the advantages of high throughput, strong distinguishability, good accuracy and high accuracy, thereby providing technical support for scientific research, fishery monitoring and ecological system investigation of the Yangtze sturgeon.

Description

Technical Field

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

[0002] The Yangtze sturgeon (Acipenser dabryanus), belonging to the Acipenseriformes order, Acipenseridae family, and Acipenser genus, is primarily found in the main and tributary rivers of the Yangtze River. It is a large, sedentary freshwater fish endemic to China. Due to human impacts, the wild population of the Yangtze sturgeon has declined sharply, and it was listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora in 2020. Surveys and restoration efforts for the wild population of the Yangtze sturgeon are urgently needed. Compared to traditional fishing methods, environmental DNA technology is relatively simple to use, and its water sampling method is more conducive to large-scale or frequent monitoring without causing 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 survey of single fish species, and the other is the survey of fish diversity. Current environmental DNA technologies are all based on mitochondrial DNA. For example, COI, 16S, 12S, or CytB fragments are used to design specific primers for single fish species or universal primers that can be used for fish diversity analysis. These are then combined with qPCR, ddPCR, or high-throughput sequencing to complete single fish species surveys or new fish diversity analyses. Because DNA is easily degraded in natural water bodies, the target fragments amplified by single primers such as COI, 16S, 12S, and CytB are generally set at 100-300bp. The short target fragments make it difficult to distinguish closely related fish species using this technology. In addition, some rare, endangered, or naturally small fish species release low concentrations of DNA into the water, which leads to the filtering of sequence information during environmental DNA monitoring, often resulting in false negatives. In short, the low DNA content and easy fragmentation of water bodies can lead to problems with 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 present invention aims to provide a primer combination, a kit, and their applications for detecting MNP marker sites in Yangtze sturgeon (Changjiang sturgeon), as well as a method for species identification based on aquatic DNA. This invention not only meets the needs for species identification and individual identification of Yangtze sturgeon, but also enables species identification and genetic diversity analysis based on aquatic DNA, with the advantages of high throughput, strong discrimination, good precision, and high accuracy.

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

[0009] The present invention provides an application of the primer composition or the kit in identifying individual Yangtze sturgeons.

[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 above-mentioned primer combination or the above-mentioned kit in the 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, multiplex PCR amplification is performed using the above primer combination or the above kit to obtain multiplex PCR amplification products;

[0015] High-throughput sequencing was performed on the multiplex PCR amplification products 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 included: 5 μL of aqueous environmental DNA, 4 μL of the above-mentioned primer combination or the above-mentioned kit, 10 μL of GenoPlexs 3×T Master Mix, and 11 μL of ddH2O;

[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 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 4 minutes, for a total of 18 cycles; extension at 72°C for 4 minutes;

[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 applications thereof for detecting MNP marker sites in Yangtze sturgeon. The primer composition comprises 100 primer pairs, the nucleotide sequences of which are shown in SEQ ID NO: 1 to SEQ ID NO: 200. Using this primer composition, not only can Yangtze sturgeon species or individuals be identified, but also Yangtze sturgeon species or genetic diversity in waters can be identified based on aquatic environmental DNA. This method has the advantages of high throughput, strong discrimination, good precision, and high accuracy, providing technical support for scientific research on Yangtze sturgeon, fishery monitoring, and ecosystem surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a genetic cluster analysis diagram of the Yangtze River sturgeon and other sturgeons in Example 3;

[0023] Figure 2 This is a distribution diagram of the difference ratio of MNP marker sites among 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 embodiments 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 embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0025] There are multiple SNP sites in the genome, and multiple nucleotide polymorphism (MNP) marker technology can combine the alleles of these different SNP sites, thereby distinguishing different individual DNA. MNP marker technology is a recently emerging method for plant variety identification after 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 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 repetitive 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 in Yangtze sturgeon, comprising 100 primer pairs, the nucleotide sequences of the 100 primer pairs are shown in SEQ ID NO: 1-SEQ ID NO: 200.

[0027] The present invention addresses the difficulties of using environmental DNA (eDNA) in fish identification and develops a primer combination for detecting the MNP marker site of the Yangtze River sturgeon. The primer combination can specifically amplify the Yangtze River sturgeon MNP marker site, 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 throughput, strong discrimination, good precision, and high accuracy. It can not only identify Yangtze River sturgeon species or individuals, but also identify Yangtze River sturgeon species or genetic diversity in water areas based on water environmental DNA. The present invention provides technical support for research such as individual tracking of Yangtze River sturgeon and release effect evaluation, helps to comprehensively evaluate the ecological status of Yangtze River sturgeon in the entire aquatic ecosystem, solves the industry problem that environmental DNA technology is difficult to distinguish closely related fish species, broadens the scope of application of environmental DNA, and also provides research data for the application of MNP marker technology in animal identification.

[0028] A second aspect of the present invention provides a kit for detecting MNP marker sites in 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 technical solution, the kit further comprises at least one of water, multiplex PCR buffer, dNTPs, and DNA polymerase.

[0031] The water can provide an ionic environment and water molecules for the multiplex PCR reaction, so that the interaction between various molecules in the reaction can be smoothly carried out, and can also help to maintain the temperature stability of the reaction system, so as to ensure the reliability and accuracy of the reaction. 2+ The multiplex PCR buffer can adjust the pH value of the reaction system, so that the action environment of the DNA polymerase is maintained to be alkaline, and the Mg

[0032] The third aspect of the present application provides application of the primer composition or the kit in Yangtze sturgeon species identification.

[0033] In a specific embodiment, the Yangtze sturgeon species identification first needs to obtain the genomic DNA of the sample to be detected, and then uses the primer composition or the kit to perform the first round of multiplex PCR amplification (the number of cycles is not more than 25) on the genomic DNA, then purifies the amplification product of the multiplex PCR, adds a sequencing adapter to the amplification product and performs the second round of multiplex PCR amplification (the number of cycles is not more than 10), to obtain the high-throughput sequencing library of the sample to be detected, and purifies and sequences the high-throughput sequencing library to obtain the sequencing data. Comparing the sequencing data with the reference sequence of the Yangtze sturgeon can obtain the sequence of the sample to be detected at the MNP marker site of the Yangtze sturgeon, so as to analyze the number of detected sites and the genotype data of the sample to be detected at the MNP site. Finally, genetic clustering analysis of the number of detected sites and the genotype data of the sample to be detected at the MNP site can determine whether the sample to be detected is a Yangtze sturgeon or other sturgeons (such as the little sturgeon and the Chinese sturgeon).

[0034] The fourth aspect of the present application provides application of the primer composition or the kit in Yangtze sturgeon individual identification.

[0035] In a specific embodiment, the Yangtze sturgeon individual identification needs to obtain the number of detected sites and the genotype data of the sample to be detected at the MNP site, compare the data with the number of detected sites and the genotype data of the candidate Yangtze sturgeon sample at the MNP site, and calculate the genetic similarity. When the genetic similarity between the sample to be detected and the candidate Yangtze sturgeon sample is ≥ 99%, the sample to be detected and the candidate Yangtze sturgeon sample are derived from the same Yangtze sturgeon individual. The primer composition or the kit provided by the present application has strong individual distinguishing ability for the Yangtze sturgeon, and is very suitable for individual identification of the Yangtze sturgeon.

[0036] A fifth 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 based on water environment DNA.

[0037] Among them, water environment DNA refers to a mixed DNA sample isolated from a water environment sample 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 carrying out 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 analyzing and calculating the above-mentioned sequence to obtain the average allele type number. The high or low average allele type 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, multiplex PCR amplification is performed using the above primer combination or the above kit to obtain multiplex PCR amplification products;

[0043] High-throughput sequencing was performed on the multiplex PCR amplification products 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 samples, and those skilled in the art can perform the extraction according to conventional methods. In a specific embodiment, the extraction can be performed using a universal DNA extraction kit for environmental samples, product number TD430-50, produced by Jianshi Biological Company.

[0045] Secondly, multiplex PCR amplification is performed using the water environment DNA as a template using the above primer combination or the above kit, with the cycle number not exceeding 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 for the first round of multiplex PCR amplification includes: 5 μL of water environment DNA, 4 μL of the above-mentioned primer composition or the above-mentioned kit, 10 μL of GenoPlexs3×T Master Mix, and 11 μL of ddH2O. Among them, 4 μL of the above-mentioned primer composition or the above-mentioned kit can be configured by the following method: 5 μL (0.2 μM) of each primer of 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 multiplex 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, for a total of 18 cycles; and extension at 72°C for 4 minutes.

[0048] 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-labeled sequences enriched in the first-round multiplex PCR through a second-round PCR amplification. The number of cycles is no more than 10, and the obtained amplified products can be used as high-throughput sequencing libraries.

[0049] Specifically, the second-round multiplex PCR amplification reaction system included: 16 μL of purified multiplex PCR product, 4 μL of 5 μM Illumina sequencing adapters, and 10 μL of GenoPlexs 3×T Master Mix. The second-round multiplex PCR amplification reaction procedure included: initial denaturation at 95°C for 3 minutes; 8 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 70°C for 30 seconds; and a final extension at 72°C for 5 minutes.

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

[0051] Specifically, the primer-marked sites of the water environment DNA can be compared with the reference sequence of the Yangtze sturgeon (the Yangtze sturgeon MNP marker site). This can obtain the sequence of the water environment sample at the Yangtze sturgeon MNP marker site, and then analyze the number of detection sites at the MNP site of the water environment sample. Finally, based on the number of detection sites at the MNP site of the water environment sample, it can be analyzed whether the Yangtze sturgeon species is present in the water area to be tested.

[0052] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

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

[0054] DNA was extracted from 30 Yangtze sturgeons obtained from the Chinese Sturgeon Research Institute of China Three Gorges Corporation to obtain the samples to be tested. Simplified genome sequencing was then performed on these samples to obtain sequencing data for each sample. Sequencing data from these samples were analyzed using Samtools (version 1.2) and BCFtools (version 1.2) sequence analysis software. Sequencing data from these samples were aligned with the reference gene sequence (SRR15851056) to identify SNPs in the Yangtze sturgeon. Alignment results were saved in the SAM (Sequence Alignment / Map) format. The SNP site was compared with the Nucleotide Sequence Database (NT) of the National Center of Biotechnology Information (NCBI) in the United States. The Yangtze sturgeon MNP marker sites were 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 between 100 and 300 bp. After screening the candidate Yangtze sturgeon MNP marker sites, the discrimination of the candidate Yangtze sturgeon MNP marker sites was analyzed using the sequencing data of the above-mentioned samples to be tested, and finally 100 Yangtze sturgeon MNP marker sites were obtained.

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

[0062] In this example, the 30 Yangtze sturgeons in Example 1 above 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: DNA samples were obtained from the tissues of the above-mentioned evaluation samples using the Animal Tissue Genomic DNA Extraction Kit (Cat. No. DP324) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. 1 μL of the DNA sample was taken from each sample to determine the DNA concentration using a Qubit fluorescence quantifier to ensure 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 ddH2O. The multiplex PCR amplification procedure includes pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 4 minutes, for a total of 18 cycles; and finally extension at 72°C for 4 minutes. After the reaction is completed, the multiplex PCR amplification products are 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 Biotechnology 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) through 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 product number N411 produced by Nanjing Novozymes Biotechnology Co., Ltd. to obtain a purified high-throughput sequencing library.

[0068] (6) Sequencing: The purified high-throughput sequencing library obtained in step (5) was sequenced using an Illumina NextSeq1000 sequencer. The detailed sequencing steps were performed according to the instructions of the sequencer. Sequencing data were obtained after sequencing was 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 tag sequences at 100 Yangtze sturgeon MNP marker sites in each of the 30 evaluation samples were aligned with the reference sequence, for a total of 3,000 Yangtze sturgeon MNP marker sites. 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 Bowtie2 software (≥90%), the Yangtze sturgeon MNP marker site was determined to be detected in the evaluation sample.

[0071] The number of detected MNP marker sites in 100 Yangtze sturgeon samples was counted in 30 evaluation samples. All 100 MNP marker sites were detected in each evaluation sample, for a detection rate of 100%. These experimental results demonstrate that the MNP marker-based detection method is highly efficient, capable of detecting up to 3,000 marker sites in a single experiment, and 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 accuracy (r) is as follows:

[0073] r = M1 / M2 × 100%, where M1 is the number of marker loci detected in both library 1 and library 2 with no genotype differences, and M2 is the number of marker loci detected in 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 River 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 sterlet sturgeon was numbered MY-1, the Chinese acipenser 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) to (7) of Example 2 to obtain the primer tag sequence of the sturgeon sample at the Yangtze sturgeon MNP marker site. Genetic cluster analysis was performed based on the sequence of the sturgeon sample at the Yangtze sturgeon MNP marker site. The specific results can be seen in FIG. Figure 1 , Figure 1 Height represents genetic distance.

[0081] like Figure 1 As shown, the Yangtze sturgeon clustered into different categories from the sterlet sturgeon and the Chinese sturgeon, and the six Yangtze sturgeon samples were significantly clustered into one category. The above results show that the 100 Yangtze sturgeon MNP marker loci 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] One sample was randomly selected from MY-3, MY-4, MY-5, MY-6, MY-7, and MY-8 in Example 3 as a sample to be tested, named MY-9, and MY-3, MY-4, MY-5, MY-6, MY-7, and MY-8 were used as a control group. MY-9 was tested according to steps (1) to (7) of Example 2 to obtain the primer tag sequence of MY-9 at the MNP marker site of the Yangtze sturgeon. The primer tag 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 sequence differences at the MNP marker site was counted. The genetic similarity between the samples was calculated and the identification conclusion was obtained. The specific results can be seen in Table 3, where the genetic similarity was calculated as follows: V1 / V2×100%, V1 is the number of marker sites detected in common with the sample to be tested and the control group, and the genotype has no difference, and V2 is the number of marker sites detected in common with the sample to be tested 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, MY-9 and MY-5 share 100% genetic similarity, indicating they are the same individual. This is consistent with the fact that the MY-9 and MY-5 samples originated from the same Yangtze sturgeon. However, the genetic differences between MY-9 and the other samples were significantly less than 99%, indicating they are different individuals. These results demonstrate that the 100 Yangtze sturgeon MNP marker loci provided by the present invention can effectively distinguish different Yangtze sturgeon individuals and can therefore be used for identification of Yangtze sturgeon individuals.

[0087] In addition, the sequencing data of the 6 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 used as the 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 used as the experimental groups. DNA was extracted from the filtered experimental group using a universal DNA extraction kit for environmental samples (product number TD430-50) produced by Jianshi Biological Company and tested according to the detection method of steps (2) to (7) of Example 2. The positive control group was tested according to the detection method of steps (1) to (8) of Example 2. The test results are shown in Table 4.

[0091] Table 4

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

[0093] As shown in Table 4, 100 MNP-labeled sites for Yangtze sturgeon were detected in the positive control group, while 61 and 47 sites were detected in the experimental groups, respectively. This indicates that Yangtze sturgeon species were present in the waters corresponding to sample 1 and sample 2, both of which are in the region of Yangtze sturgeon occurrence in Yibin City. These results are consistent with the actual situation and demonstrate that the MNP-labeled detection method can be used to identify Yangtze sturgeon species using aquatic DNA as a sample.

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

[0095] The water environment sample obtained from the downstream of Xiangjia Dam on the Jinsha River in Yibin City was named BX-R2, the water environment sample obtained from the Chishui River estuary in Hejiang County was named HJ2-R3, and the water environment sample obtained from the downstream of Luzhou was named T1-L2. BX-R2, HJ2-R3, and T1-L2 were filtered to obtain samples to be tested. DNA was extracted using the universal DNA extraction kit for environmental samples, product number TD430-50, produced by Jianshi Biological Company. Subsequently, the test was performed according to the detection method of steps (2) to (8) of Example 2. The test results are shown in Table 5, where the average allele number (Gd) of the MNP marker is calculated 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 greater genetic diversity in fish species is associated with a greater number of alleles, measuring the average number of alleles in aquatic samples can reflect the genetic diversity of Yangtze sturgeon in the corresponding waters. As shown in Table 5, the average number of alleles at the BX-R2 MNP marker locus was higher than in other samples, indicating that the genetic diversity of Yangtze sturgeon in the area downstream of Xiangjiaba Dam in the Jinsha River, Yibin City, is higher than in other areas. These results demonstrate that the MNP-based detection method can be used to analyze the abundance and genetic diversity of Yangtze sturgeon in waters using aquatic DNA as a sample.

[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 mine the MNP site that is different from other species. Then, the conserved sequences on both sides of the MNP site are used to design MNP site detection primers suitable for multiplex PCR amplification. Subsequently, based on the test results of the standard, a set of MNP sites with high polymorphism and strong specificity and the most compatible primer composition and kit are screened. The present invention uses multiplex PCR amplification technology and second-generation sequencing technology through the primer composition and kit to identify the MNP marker site of the Yangtze sturgeon. It 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 surveys 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 they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

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

200.

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

3. The kit according to claim 2, wherein 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 species identification of Yangtze sturgeon.

5. Use of the primer composition according to claim 1 or the kit according to claim 2 or 3 in identifying individual Yangtze sturgeons.

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

7. Use of the primer combination 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 River 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 to obtain water environment DNA; Using the water environment DNA as a template, performing multiplex PCR amplification using the primer composition of claim 1 or the kit of claim 2 or 3 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 water area 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 for the first round of multiplex PCR amplification includes: 5 μL of the water environment DNA, 4 μL of the primer combination 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 included: 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 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.

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