Fish DNA bar code database and mitochondrial genome-based complete sequence construction method and application thereof

Abstract

The invention relates to the field of molecular biology, and discloses a fish DNA bar code database and a construction method and application thereof based on a mitochondrial genome complete sequence. The method comprises the steps of fish directory determination, fish collection, classification and identification, DNA extraction, mitochondrial whole genome sequencing and analysis, DNA bar code database building and database application. Based on the research basis of the target water area and fish historical data, the fish directory in the target water area is determined, the fish DNA bar code database is constructed by utilizing the mitochondrial whole genome sequencing technology, and accurate database support is provided for fish investigation and monitoring by applying the environment DNA technology. The problems that an existing fish public database is insufficient in integrity, miscellaneous in data source, uneven in quality and insufficient in region pertinence, possibly causes small population species comparison loss, comparison results are wrong, false positive results such as non-target region species cannot be effectively eliminated, and related species with high gene similarity are difficult to accurately identify are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a fish DNA barcode database and a construction method and application thereof based on the complete sequence of a mitochondrial genome. Background Art

[0002] Fish are an important part of aquatic ecosystems, and their species composition and genetic diversity are important indicators of the health of aquatic ecosystems. Water pollution, habitat fragmentation, and invasion of alien species have led to the decline of fish resources and imbalanced community structure. By conducting fish resource surveys and monitoring, we can understand the species composition and diversity of fish in target waters, understand the status of rare and endangered fish populations, and support species protection, resource conservation, and habitat restoration.

[0003] At present, fish resource surveys and monitoring mostly use nets to capture fish samples. The fishing process requires a lot of manpower, causes a certain degree of damage to fish resources, and also disturbs fish habitats. From the analysis of survey and monitoring results, net fishing is not ideal for catching fish with small populations, small individuals, and high concealment, and has high requirements for the classification and identification level of investigators. Environmental DNA technology has the characteristics of non-destructive sampling, large-scale continuous sampling, high precision, and low cost, and has gradually been widely used in fish resource surveys and monitoring.

[0004] DNA barcode is the core component of environmental DNA technology. DNA barcode refers to a short DNA fragment that can correspond to species, is relatively conservative and easy to amplify, and can be used for species identification. The mitochondrial genome has the characteristics of rapid evolution, maternal inheritance, lack of recombination, etc. It contains a high copy number and many conservative sequences that can be used to distinguish species. It is a good material for DNA barcode. Commonly used mitochondrial genes include Cytb, COI, 12S, 16S, D-loop, etc., with a fragment size of 80-200bp. The design of universal primers for fish usually selects mitochondrial gene fragments such as Cytb, 12S, and COI, among which the 12S gene is the most widely used. When using primers to amplify gene sequences by PCR, errors will occur due to the formation of chimeras, heteroduplex molecules, and Taq DNA polymerase errors, so the effect of sequence amplification is greatly affected by the efficiency of primers. The amplified sequence is compared with the DNA barcode, and the taxonomic status of the species can be determined based on the sequence overlap rate. For some closely related species with a short differentiation time, it will be difficult to distinguish. The mitochondrial whole genome sequencing process does not involve the PCR step, which can effectively avoid errors caused by the amplification process; at the same time, the sequence obtained by whole genome sequencing is complete, and using it for sequence comparison can make up for the deficiency of local fragment comparison that cannot distinguish closely related species.

[0005] The construction of barcode database is the basis for the implementation of environmental DNA technology. The National Center for Biotechnology Information (NCBI) and CBOL established the first database system GenBank in 1982. Later, the Consortium for the Barcode of Life (CBOL) established the Barcode of Life Database System BOLD (Barcode of Life Data Systems) in 2004. In 2007, Canada established the DNA Barcode Identification Center. DNA barcode technology compares the target sequence used for species identification with the sequence in the database to determine the species information. It has high requirements for the richness and accuracy of the database. If the DNA sequence of a species is not included in the database or the amount of data included is small or wrong, the comparison will fail and the comparison result will be wrong. At present, the comprehensiveness of species and the accuracy of sequences in public databases cannot meet the needs of specific research. There are cases where the gene sequences of unique species in the target waters are not included, and the accuracy of the included sequences needs to be verified. Therefore, it has become a trend to build an accurate and traceable self-built database according to research needs.

[0006] The length of fish mitochondrial genomes is mostly around 15-20kb, which is much longer than the length of common DNA barcodes. Therefore, if the full sequence of fish mitochondrial genomes can be used as fish DNA barcodes, a wider range of sequence differences can be identified, and the accuracy of species identification will be greatly improved. The method of using the full sequence of mitochondrial genomes to construct a fish DNA barcode database can effectively improve the accuracy of environmental DNA species comparison results, simplify the technical steps, and is more conducive to the development of environmental DNA monitoring technology. Summary of the invention

[0007] Technical problems to be solved: In view of the technical problems that the existing public fish databases are not complete enough, the data sources are complex and the quality is uneven, and the regional specificity is insufficient, which may lead to the loss of small population species comparison, erroneous comparison results and the inability to effectively exclude false positive results such as species in non-target areas, and the difficulty in accurately identifying closely related species with high genetic similarity, the present invention provides a fish DNA barcode database and a construction method and application based on the full sequence of mitochondrial genome. The fish barcode database is constructed using the full mitochondrial genome, which is simple to operate and highly feasible.

[0008] Technical solution: A method for constructing a fish DNA barcode database based on the complete sequence of the mitochondrial genome, the steps are as follows:

[0009] (1) Determine the fish list;

[0010] (2) Collect and identify samples according to the fish list and preserve the specimens;

[0011] (3) Extract DNA from the specimen;

[0012] (4) Sequencing and analyzing the entire mitochondrial genome of the extracted DNA samples;

[0013] (5) Construct a DNA barcode library.

[0014] Preferably, when determining the fish list in step (1), the fish list is determined based on the survey basis of the target waters and historical fish data.

[0015] Preferably, in step (2), samples are collected and identified according to the fish list, and specimens are preserved, specifically as follows: samples are collected according to the fish list, species are identified based on taxonomic basis, morphologically intact individuals of each species are selected to take photos of morphological characteristics, and muscle samples are collected and made into specimens for preservation.

[0016] Furthermore, fish individuals in the target waters are collected first. If the number of fish individuals collected in the target waters is insufficient, individuals from adjacent waters are used to supplement the collection.

[0017] Preferably, in step (3), DNA is extracted from the sample by using SDS extraction combined with purification column method.

[0018] Preferably, the extraction method is as follows: dorsal muscles are taken from three individuals of each fish species, and total DNA is extracted by SDS (sodium dodecyl sulfate) extraction combined with purification column method. 500 mg of muscle is taken from each sample, and lysed using SDS lysis buffer, and appropriate amounts of proteinase K and mercaptoethanol are added. During the lysis process, the muscles are gently inverted and mixed, and the muscles are centrifuged after cooling at room temperature. The supernatant of the centrifugation is extracted by adding chloroform / isoamyl alcohol (24:1), and the extraction is performed twice. DNA is precipitated with isopropanol, and the muscles are slightly inverted and mixed before centrifugation. The waste liquid is discarded, and the precipitate is washed with 75% ethanol, and the reaction is repeated twice. After the DNA precipitate is dried, 200 ul TE (Tris-EDTA) is added to dissolve the precipitate, and the RNase is digested. The OMEGA purification column is used for purification, and after the purification is completed, the Ampure XPbeads kit is used for purification. After purification, Nanodrop, Qbuit and electrophoresis quality inspection are performed.

[0019] Preferably, in step (4), the extracted DNA sample is subjected to mitochondrial whole genome sequencing and analysis, specifically as follows: the sample genomic DNA is broken into 300-500 bp fragments using a Covaris ultrasonic disruptor, the DNA fragment ends are repaired, and an A base and a sequencing adapter are added to the 3′ end; the ligation product is amplified by PCR, the product is recovered and purified using magnetic beads, and a library is constructed; after the library construction is completed, a preliminary quantification is first performed using Qubit 3.0, the library is diluted, and then the insert fragment of the library is detected using Agilent 2100. After the insert fragment size meets the expectation, the effective concentration of the library is accurately quantified using the Q-PCR method; after the DNA library passes the quality inspection, paired end sequencing is performed using the MGISEQ-T7 high-throughput sequencing platform; the sequencing data is quality controlled and filtered, and the UPARSE software (http: / / drive5.com / uparse / , version 3.0) is used to analyze the insert fragments of the library. 7.1), the sequences were clustered into OTUs according to 97% similarity and chimeras were removed. After excluding erroneous sequences, the data yield, base content distribution, and data quality distribution were calculated, and the mitochondrial genome was assembled and annotated.

[0020] Preferably, the entry of the DNA barcode in step (5) includes at least one of the following information: Chinese name of the species, Latin name, taxonomic status, mitochondrial genome sequence, morphological picture, specimen, muscle sample, DNA sample, latitude and longitude of the sampling point and sampling time.

[0021] A fish DNA barcode database constructed by the above method.

[0022] Based on the above-mentioned fish DNA barcode database, it is used to determine the taxonomic level and relative abundance of fish species in target waters.

[0023] Preferably, the steps are as follows: collect water samples in the target waters, use microporous filter membranes and vacuum filtration devices to enrich DNA in the water samples to obtain environmental DNA samples, perform full-sequence short-read sequencing on the experimental samples, compare the sequencing results using BWA software based on the above-mentioned DNA barcode database, directly count the reads of specific alignments, classify the reads of multiple alignments according to the taxonomic units of the alignment sequences, obtain species annotations through the taxonomic information corresponding to the database as a whole, and determine the taxonomic level and relative abundance of fish species in the target waters.

[0024] Beneficial effects:

[0025] (1) The fish list contained in the database provided by the present invention is based on the research foundation and historical data collection of the target waters, which effectively eliminates false positives in the comparison results and improves the credibility of the comparison results.

[0026] (2) The present invention uses mitochondrial whole genome sequence comparison to make up for the deficiency that local fragment comparison cannot distinguish closely related species.

[0027] (3) The present invention avoids the PCR amplification step required by conventional methods, shortens the technical process, simplifies the sequencing steps, and avoids the situation where species cannot be detected due to primer mismatch, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis result diagram of the fish mitochondrial genome in Example 1 of the present invention;

[0029] Figure 2 A photograph of a fish in which one of the morphological species is identified as Coilia edulis in Example 1 of the present invention;

[0030] Figure 3 The entry information of one of the Coilia edulis in the database of Example 1 of the present invention;

[0031] Figure 4 This is a sequence alignment process diagram of Example 2 of the present invention. DETAILED DESCRIPTION

[0032] The present invention aims at the actual needs of fish resource survey and monitoring in the field of ecological environment protection, and uses molecular biology methods to construct a fish DNA barcode database. The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The implementation cases described below are only part of the embodiments of the present invention. Based on this, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present invention.

[0033] Unless otherwise specified, the raw materials and instruments used in the examples of the present invention are common commercially available products.

[0034] The specific implementation cases are as follows:

[0035] Example 1

[0036] Establishing a fish barcode database in the Jiangsu section of the Yangtze River

[0037] (1) Determine the fish list: Collect historical data on fish in the target waters (in this example, refer to the Yangtze River Fish Records, Jiangsu Fish Records, and historical survey results of the lower reaches of the Yangtze River from 2017 to the sampling time) to determine the fish list in the lower reaches of the Yangtze River.

[0038] (2) Fish collection and classification and identification: Samples are collected according to the fish list, with priority given to fish individuals in the Jiangsu section of the Yangtze River. If the number of fish collected in the Jiangsu section of the Yangtze River is insufficient, individuals from neighboring waters are used as supplements. Species are identified based on taxonomic basis, and 3 individuals with intact morphology are selected for each species to take photos of their morphological characteristics, and specimens are prepared for preservation. This example takes Coilia scaly as an example, and its morphological characteristics photos are taken as shown in Figure 2 .

[0039] (3) DNA extraction: Dorsal muscles were taken from three individuals of each fish species, and DNA was extracted by SDS (sodium dodecyl sulfate) extraction combined with purification column method. 500 mg of muscle was taken from each sample and lysed with 700 ul SDS lysis buffer, 10 ul proteinase K and 10 ul mercaptoethanol were added. During the lysis process, the muscles were gently inverted to mix well. After cooling at room temperature, the muscles were centrifuged at 12000 rpm for 5 min. The supernatant was extracted with chloroform / isoamyl alcohol (24:1). The extraction was repeated twice. DNA was precipitated with isopropanol, gently inverted to mix well, and then centrifuged. The waste liquid was discarded, and 800 μL 75% ethanol was added to wash the precipitate. The DNA precipitate was dried and dissolved with 200 ul TE (Tris-EDTA). 30 ul RNAse digestion was performed and purified using purification columns (OMEGA). After purification, the Ampure XPbeads kit was used for purification. After purification, Nanodrop and Qbuit and electrophoresis quality inspection were performed (see Figure 1 ).

[0040] (4) Mitochondrial whole genome sequencing and analysis: The extracted DNA samples were used for mitochondrial whole genome sequencing. The sample genomic DNA was broken into 300-500 bp fragments using a Covaris ultrasonic disruptor. The DNA fragments were repaired at the ends, and the A base and sequencing adapter were added to the 3′ end. The ligation products were amplified by PCR. The reaction system was 20 μl product, 25 μl VAHTS HiFi Amplification Mix, 5 μl PCR Primers. The reaction procedure was 95°C for 3 min, 98°C for 20 sec, 60°C for 15 sec, 72°C for 30 sec for 6 cycles, 72°C for 5 min, and stored at 4°C. The products were recovered and purified using magnetic beads, and the library was constructed. After the library was constructed, Qubit 3.0 was used for preliminary quantification and the library was diluted. Then, the insert fragments of the library were detected using Agilent 2100. The insert fragment size was 400-600 bp, and the effective concentration of the library was accurately quantified using the Q-PCR method. After the DNA library was qualified, paired end sequencing was performed using the MGISEQ-T7 high-throughput sequencing platform. The sequencing data was quality-controlled and filtered, and the sequences were clustered into OTUs and chimeras were removed based on 97% similarity using UPARSE software (http: / / drive5.com / uparse / , version 7.1). After excluding erroneous sequences, the data yield, base content distribution, and data quality distribution were counted, and the mitochondrial genome was assembled and annotated.

[0041] (5) DNA barcode database construction: The information of the three individuals collected from each species is sorted and summarized to establish a standardized species barcode database entry format including the species' Chinese name, Latin scientific name, taxonomic status, mitochondrial genome sequence, morphological pictures, specimens, muscle samples, DNA samples, latitude and longitude of the sampling point, sampling time, etc., and to establish a fish barcode database for the Jiangsu section of the Yangtze River. This example takes Coilia scabra as an example, and its entry information in the database is shown in Figure 3 Due to limited space, the DNA barcode in the figure is specifically the sequence of SEQ ID NO: 1 + SEQ ID NO: 2. SEQ ID NO: 1 and SEQ ID NO: 2 can be found in the sequence listing.

[0042] Example 2

[0043] Species identification of environmental DNA samples using the fish barcode database from the Jiangsu section of the Yangtze River

[0044] (1) Obtaining environmental DNA samples: Based on the principles of uniformity and representativeness, a sampling section was set up every 20 km in the Jiangsu section of the Yangtze River, with a total of 18 sampling sections. The surface water about 0.5 m underwater was collected using a disinfected water sample collector and placed in a 2L sampling bottle. The water was stored in a dark and low-temperature environment. Six water samples were collected evenly from the north bank to the south bank of each section. The water samples were filtered using a vacuum filtration pump and a 0.45 μm mixed cellulose microporous filter membrane to enrich the environmental DNA in the water samples. After the water samples were filtered, the filter membrane was placed in a 5 mL sample tube filled with anhydrous ethanol and stored at -20°C.

[0045] (2) DNA extraction: Extract membrane DNA. The extraction steps are the same as those in (3) of Example 1 (replacing muscle with membrane DNA in this example).

[0046] (3) Mitochondrial whole genome sequencing and analysis: The extracted environmental DNA samples were subjected to mitochondrial whole-sequence short-read sequencing, and the sequencing steps were the same as (4) of Example 1 to obtain genomic DNA.

[0047] (4) Library construction: The library was constructed using the MGIEasy Universal DNA Library Preparation Kit V1.0 (CAT#1000005250, MGI). The steps are as follows: 1 μg of genomic DNA was randomly fragmented by Covaris, and DNA fragments with an average size of 200-400 bp were selected. Afterwards, the selected fragments were end-repaired, 3' olefinated, and then connected to the adapter. The DNA sample was amplified by PCR and further purified. The double-stranded PCR product was heat denatured and cyclized. The single-stranded circular DNA (ssCir-DNA) was formatted into the final library and identified by QC. Qualified libraries were sequenced by PE150 on the DNBSEQ-T7RS platform.

[0048] (5) Species comparison: see Figure 4 For the short reads obtained by sequencing, they are aligned back to the aforementioned "Fish Barcode Database of Jiangsu Section of the Yangtze River" through BWA to screen high-quality alignment results. For reads that can specifically align back to a unique sequence, they are assigned to the species corresponding to the sequence; for reads with multiple alignments, the species corresponding to the aligned sequences are classified to the genus / family / order level. Finally, the abundance of each species in each sample is determined. By normalizing and correcting the results of multi-sample alignments, the abundance of each species in each sample in the database is obtained. Through the above data, the samples are further clustered to characterize the similarity relationship between the samples to the included species; the species are clustered to characterize the distribution similarity relationship of different species in the above samples. .

[0049] Finally, it should be noted that the above implementation modes are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned implementation modes, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned implementation modes can still be modified, or some or all of the technical features therein 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 implementation modes of the present invention.

Claims

1. A method for constructing a fish DNA barcode database based on the complete sequence of the mitochondrial genome, characterized in that: Here are the steps: (1) Determine the fish list; (2) Collect and identify samples according to the fish list and preserve the specimens; (3) Extract DNA from the specimen; (4) Sequencing and analyzing the entire mitochondrial genome of the extracted DNA samples; (5) Construct a DNA barcode library.

2. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 1, characterized in that: When determining the fish list in step (1), the fish list is determined based on the survey basis of the target waters and historical fish data.

3. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 1, characterized in that: In the step (2), samples are collected and identified according to the fish list, and specimens are preserved, specifically as follows: samples are collected according to the fish list, species are identified according to taxonomic basis, morphologically intact individuals of each species are selected to take photos of morphological characteristics, and muscle samples are collected and made into specimens for preservation.

4. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 1, characterized in that: In the step (3), DNA is extracted from the sample by using SDS extraction combined with purification column method.

5. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 4, characterized in that: The extraction method is specifically as follows: dorsal muscles are taken from three individuals of each fish species, lysed using SDS lysis solution, proteinase K and mercaptoethanol are added, the muscles are gently inverted and mixed during the lysis process, the muscles are cooled at room temperature and then centrifuged, the supernatant of the centrifugation is taken and chloroform / isoamyl alcohol is added for extraction, the extraction is performed twice, DNA is precipitated with isopropanol, the muscles are gently inverted and mixed and then centrifuged, the waste liquid is discarded, 75% ethanol is added to wash the precipitate, and the process is repeated twice, the DNA precipitate is dried and then TE is added to dissolve it, RNAse digestion is performed, and purification is performed using a purification column, and after purification, an Ampure XP beads kit is used for purification, and Nanodrop, Qbuit and electrophoresis quality inspection are performed after purification, and the qualified ones are accepted.

6. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 1, characterized in that: In the step (4), the extracted DNA sample is subjected to mitochondrial whole genome sequencing and analysis, specifically as follows: the sample genomic DNA is broken into 300-500 bp fragments using a Covaris ultrasonic disruptor, the DNA fragment ends are repaired, and an A base and a sequencing adapter are added to the 3′ end; the ligation product is amplified by PCR, the product is recovered and purified using magnetic beads, and a library is constructed; after the library construction is completed, a preliminary quantification is first performed using Qubit 3.0, the library is diluted, and then the insert fragment of the library is detected using Agilent 2100. After the insert fragment size meets the expectation, the effective concentration of the library is accurately quantified using the Q-PCR method; after the DNA library passes the quality inspection, double-end sequencing is performed using the MGISEQ-T7 high-throughput sequencing platform; The sequencing data were quality-controlled and filtered. UPARSE software was used to cluster the sequences into OTUs based on 97% similarity and remove chimeras. After excluding erroneous sequences, the data yield, base content distribution, and data quality distribution were analyzed, and the mitochondrial genome was assembled and annotated.

7. The method for constructing a fish DNA barcode database based on the complete mitochondrial genome sequence according to claim 1, characterized in that: The entry of the DNA barcode in step (5) includes at least one of the following information: Chinese name of the species, Latin name, taxonomic status, mitochondrial genome sequence, morphological picture, specimen, muscle sample, DNA sample, latitude and longitude of the sampling point and sampling time.

8. A fish DNA barcode database constructed according to the method of claim 1.

9. Application of the fish DNA barcode database according to claim 8 in determining the taxonomic level and relative abundance of fish species in target waters.

10. The use according to claim 9, characterized in that: The steps are as follows: collect water samples in the target waters, use a microporous filter membrane and a vacuum filtration device to enrich the DNA in the water samples, obtain environmental DNA samples, perform full-sequence short-read sequencing on the experimental samples, compare the sequencing results using BWA software based on the DNA barcode database of claim 8, directly count the reads of specific alignments, classify the reads of multiple alignments according to the taxonomic units of the alignment sequences, obtain species annotations through the taxonomic information corresponding to the database as a whole, and determine the taxonomic level and relative abundance of fish species in the target waters.

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