A kind of lingual oyster mitochondrial genome sequence and lingual oyster DNA barcode identification primer and method

Abstract

The application discloses a lingual oyster mitochondrial genome sequence and a lingual oyster DNA barcode identification primer and method. The identification primer comprises a nested peripheral primer and a nested inner primer, the nested peripheral primer comprises HSCOI OU F: 5'-AT GCGTTGGGAGTTTGTGGTA-3' and HSCOI OU R: 5'-TCGAAAGAACATAATGAAAGTGGC-3'; and the nested inner primer comprises HSCOI IN F: 5'-CTATAAACGGCACTGGATGGA-3' and HSCOI IN R: 5'-AAAGACACGTCAACAGAACCC-3'. The application provides the lingual oyster mitochondrial genome sequence, and provides the lingual oyster DNA barcode identification primer and detection method, and provides an effective method for classification and identification of marine resources.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and molecular biology, specifically relating to a mitochondrial genome sequence of the hyoid oyster and primers and methods for identifying the hyoid oyster. Background Technology

[0002] With the development of biotechnology, since each organism's genes have their own unique nucleotide sequence, nucleotide sequences carried by DNA are used as digital barcodes, similar to those on product labels. Therefore, DNA nucleotide sequences (i.e., DNA barcoding) are used as identification tags for species. Thus, establishing a database of marine organism DNA barcodes is of great significance for the development and utilization of marine biological resources.

[0003] However, no method is foolproof. While DNA barcoding offers advantages in species identification such as short sequences and fast testing speed, it also has its limitations. Because DNA classification methods rely on the frequency of genetic variation in an organism's DNA, it is difficult to classify recently diverged or new species due to the slow rate of DNA mutation and the weak changes in the short term. Furthermore, it is impractical to classify and identify all species based on a single gene. DNA barcoding identification is also based on existing, established species. Therefore, we can utilize mitochondrial genomes for accurate species classification, providing a foundation for the development of marine biological resources. The hyoid oyster is a large oyster belonging to the animal kingdom, phylum Mollusca, class Bivalvia, order Pearbilales, family Crassulaeidae, and genus Crassula. Summary of the Invention

[0004] This invention provides a mitochondrial genome sequence of the Crassula hyoides and a DNA barcoding detection method, providing an effective approach for the classification and identification of marine resources.

[0005] The first objective of this invention is to provide the mitochondrial genome sequence of the hyoid oyster, as shown in SEQ ID NO.1.

[0006] The second objective of this invention is to provide a DNA barcoding primer for Crassula hyoides, comprising nested outer primers and nested inner primers. The nested outer primers include HSCOI OU F: 5'-ATGCGTTGGGAGTTTGTGGTA-3' and HSCOI OU R: 5'-TCGAAAGAACATAATGAAAGTGGC-3'; the nested inner primers include HSCOI INF: 5'-CTATAAACGGCACTGGATGGA-3' and HSCOI IN R: 5'-AAAGACACGTCAACAGAACCC-3'.

[0007] A third objective of this invention is to provide a kit for identifying hyoid oysters containing the aforementioned DNA barcoding primers for hyoid oyster identification.

[0008] The fourth objective of this invention is to provide a method for identifying the hyoid oyster, comprising the following steps:

[0009] The mitochondrial genome of the sample to be tested is extracted, and the first PCR amplification is performed using nested outer primers to obtain the first amplification product. Using the first amplification product as a template, the second amplification is performed using nested inner primers to obtain the second amplification product. If amplification products can be obtained in both tests, and the second amplification product is 1000bp in size, then it is hyoid oyster; otherwise, it is not.

[0010] Preferably, the PCR amplification system for the first PCR amplification is: 1 μl template, 9.5 μl ultrapure water, 12.5 μl 2×Taq PCR Master-Mix, and 2 μl each of 1 mmol / L HSCOI OU F and HSCOI OU R.

[0011] Preferably, the first PCR amplification is performed using the following PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 51 ℃ for 50 s, 72 ℃ for 1.2 min, for 30 cycles; followed by an extension at 72 ℃ for 10 min.

[0012] Preferably, the second PCR amplification consists of 1 μl template, 9.5 μl ultrapure water, 12.5 μl 2×Taq PCR Master-Mix, and 2 μl each of 1 mmol / L HSCOI IN F and HSCOI IN R.

[0013] Preferably, the second PCR amplification is performed using the following PCR amplification program: 94 ℃ for 5 min; 94 ℃ for 30 s, 51 ℃ for 50 s, 72 ℃ for 1.2 min, for 30 cycles; and an extension at 72 ℃ for 10 min.

[0014] In recent years, molecular phylogenetics has become a standard method in taxonomic research. However, with the deepening of research, increasing evidence suggests that the evolutionary rates of single genes or a few genes differ between and within species, failing to fundamentally represent the evolutionary pattern of species. With continuous innovation in sequencing technology and decreasing sequencing costs, the development of mitochondrial sequencing technology has brought molecular phylogenetics into a new research field. Therefore, mitochondria can be used to study molecular evolution and explore the origin of species. The hyoid oyster is a large animal, belonging to the kingdom Animalia, phylum Mollusca, class Bivalvia, order Pyrenolochiales, family Ostreidae, and genus Crassula. Currently, there is no mitochondrial genome sequence for this species globally. This invention provides a mitochondrial genome sequence of the Crassula hyoides and a DNA barcoding identification primer and detection method, providing an effective method for the classification and identification of marine resources. Attached Figure Description

[0015] Figure 1 This document presents the mitochondrial genome sequencing flowchart and map of the hyoid oyster. A, Mitochondrial genome sequencing flowchart; B, Mitochondrial genome map of the hyoid oyster. Genes within loops represent transcription in a clockwise direction, while genes outside loops are transcribed in the opposite direction. Different functional genes are labeled with different colors. An embedded gray histogram displays the GC content of the genome, with the middle gray line representing the 50% threshold.

[0016] Figure 2This is a cloning and evolutionary classification of mitochondrial genes in the Crassula hyoides. A, the results of DNA barcoding electrophoresis of 1000 bp nucleic acid sequences obtained using nested primers; B, the evolutionary classification of the COI gene in the Crassula hyoides, compared with Crassostrea gigas, KJ855245.1; Crassostrea sikamea, FJ841966.1; Crassostrea hongkongensis, FJ841963.1; Crassostrea ariakensis, EU672835.1; Crassostrea iredalei, FJ841967.1; Saccostrea mordax, KP769562.1; Saccostrea kegaki, KT936587.1; Saccostrea cucullata, MF198445.1; Saccostrea malabonensis, ON649706.1; Neopycnodonte cochlear, In comparison with AB076939.1; Pycnodonte taniguchii, AB076916.1; Hyotissaimbricata, AB076917.1; Hyotissa hyotis, GQ166583.1; Cellana orientalis, NC_063771.1; Clithon squarrosum, NC_062613.1; Lunella correensis, MT185943.1; Lineus viridis, FJ839919.1; Nipponnemertes punctatula, KC710980.1; Viviparuschui, KY679829.1; Cultellus attenuatus, MW653805.1, this sequence clustered with Hyotissa hyotis, indicating that this sequence is part of the mitochondrial genome of Crassula hyoides. Detailed Implementation

[0017] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0018] Example 1 Sequencing procedure as follows Figure 1 As shown in A

[0019] 1. Sample collection

[0020] 1) Samples of Crassula hyoides were collected from islands and reefs in the Xisha Islands, including Zhaoshu Island, Langhua Reef, Jinqing Island, and Beidao Island, within a depth of 5-20 meters. Individual samples were obtained by diving, the shells were pried open, and approximately 0.5 grams of adductor muscle tissue was separated and stored in 5 ml of anhydrous ethanol at -20°C for later laboratory processing.

[0021] 2) Main instruments: tissue homogenizer (QIAGEN, USA); nucleic acid and protein analyzer (nanodrop one, Thermo Fisher, Germany); PCR instrument (Verititm 96 Well Thermat Cycler, Gene, USA); gel imaging analysis system (BIO-RAD, BIO-RAD, USA).

[0022] 3) Main reagent homogenate: 25 mmol / L Tris-HCl, 30 mmol / L EDTANa2, 50 mmol / L glucose (pH 8.0), 20% SDS, 20 mg / ml proteinase K, 3 mol / L potassium acetate (pH 5.4), containing 3 mol / L potassium acetate and 2 mol / L acetic acid, Tris-saturated phenol, chloroform, isoamyl alcohol, isopropanol, and 75% ethanol.

[0023] 2. Mitochondrial genome extraction

[0024] 1) Homogenization. For a single sample, take 100 mg of adductor muscle tissue, place it in a homogenizer, add 500 μl of homogenate, and homogenize the adductor muscle tissue for 2 min. Transfer the homogenate to a centrifuge tube, add another 300 μl of homogenate to the homogenizer, and homogenize the abdominal foot muscle tissue. Repeat the homogenization process 3 times. Finally, rinse the homogenizer with 100 μl of homogenate to form a washing solution, which is then transferred to a centrifuge tube.

[0025] 2) Crack. Heat the homogenate in a 100 ℃ water bath for 10 min, then cool to room temperature.

[0026] 3) Digestion. Add 80 μl SDS and 60 μl proteinase K to the homogenate and mix well. Heat to 60 °C and keep warm for 1 h; cool to 37 °C and keep warm for 1 h; heat to 60 °C again and keep warm for 1 h; cool to 37 °C and keep warm for 1 h.

[0027] 4) Purification. Add 1.2 ml of potassium acetate (pH 5.4), gently invert 8-10 times, and let stand at 4 ℃ for 30 min; centrifuge at 14000 r / min for 15 min, take 1 ml of supernatant into a clean 2.5 ml centrifuge tube, add another 1 ml of potassium acetate (pH 5.4), gently invert 8-10 times, and let stand at 4 ℃ for 30 min; take another 1 ml of supernatant into a clean 2 ml centrifuge tube, add an equal volume of potassium acetate solution, gently invert 7-8 times, and let stand at 4 ℃ for 30 min.

[0028] 5) After precipitation, centrifuge at 4 ℃, 12,000 r / min for 15 min, take 950 μl of supernatant and place them in clean 2 ml centrifuge tubes respectively; add an equal volume of isopropanol to each tube and refrigerate at -20 ℃ for 30 min.

[0029] 6) Wash and air dry at 4 ℃, centrifuge at 14000 r / min for 15 min, and remove the supernatant; add 200 μl of 75% ethanol, wash twice, air dry, add an appropriate amount of double-distilled water to dissolve, perform gel electrophoresis, and detect the band brightness; in addition, DNA content is detected at the same time to determine the solution concentration, and store at -20 ℃ for later use.

[0030] 2. Mitochondrial genome sequencing

[0031] 1) Take a 2μg mitochondrial genome sample and send it to a sequencing company (Huitong Biotechnology, China) via dry ice express for mitochondrial sequencing.

[0032] 2) Total DNA sequencing of the samples was performed using Illumina next-generation sequencing technology. An Illumina PE library was constructed, and paired-end sequencing of the sample DNA was performed using Illumina Hiseq sequencing technology.

[0033] 3) After obtaining the raw data, the data is trimmed to improve its quality. This includes removing adapter sequences from reads; removing non-AGCT bases at the 5' end; trimming the ends of reads with low sequencing quality (sequencing quality value less than Q20); removing reads containing N up to 10%; and discarding adapters and small fragments less than 50bp in length after quality trimming.

[0034] 4) The sequencing quality has been observed to be very good. Next, quality control of the sequencing data is performed using NGSQC software. The results generate a series of images and reports.

[0035] 5) Genomic annotation of sequencing organelles is mainly divided into three parts: protein-coding gene annotation; RNA annotation; and structural annotation. The Hyotissa sp sequencing data insert is 300 bp. The mitochondrial splicing results show a circular genome with a length of 24,741 bp, as shown in SEQ ID NO.1, with an average sequencing depth of 629.4X. The GC content is 40.6%, and the contents of other bases are: A 24.2%, C 13.8%, G 26.8%, T 35.2%, encoding 43 genes (…). Figure 1 B).

[0036] 3. Cloning of the DNA barcode gene COI

[0037] 1) PCR detection: CO I primers were used to amplify mitochondrial genes. Nested PCR was employed, and the primers are listed in Table 1. The PCR reaction system contained 1 μl template, 9.5 μl ultrapure water, and 12.5 μl Mix (2×Taq PCR Master-Mix, TIANGEN). Initially, 2 μl each of the outer primers HSCOI OU F and HSCOI OU R (1 mmol / L) were used. PCR reaction conditions: 94 ℃ for 5 min; 94 ℃ for 30 s, 51 ℃ for 50 s, 72 ℃ for 1.2 min, 30 cycles; extension at 72 ℃ for 10 min, incubation at 4 ℃; obtain the outer primer DNA template, dilute 1000-fold with sterile distilled water as the inner PCR DNA template, 1 μL each of inner primers HSCOI IN F and HSCOI IN R (1 mmol / L), repeat the above PCR procedure to obtain PCR products, amplified products were analyzed by 1.5% agarose gel electrophoresis, and DNA amplification bands were analyzed under UV light to obtain a target band of approximately 1 kB. Figure 2 A).

[0038] Table 1 Nested Primer Table

[0039]

[0040] 2) Purification was performed using the HiPure Gel Pure DNA Kits. The specific steps are as follows:

[0041] (1) Place the gel under a UV lamp and quickly cut the gel of the target band fragment into a 1.5 mL centrifuge tube to avoid cutting off excess and useless gel.

[0042] (2) Weigh the gel and add 100 μL of Buffer GDP for every 100 mg of gel. Incubate in a metal bath at 55 °C for 15 min, and manually invert twice to completely dissolve the gel.

[0043] (3) Briefly centrifuge and transfer ≤700μL of gel solution to the column, 12000×g for 1 min.

[0044] (4) Discard the filtrate, put the column back into the collection tube, add 300 μL of Buffer GDP into the column, let it stand at room temperature for 1 min, and then 12000×g for 1 min.

[0045] (5) Discard the filtrate, add 600 μL Buffer DW2 (diluted with anhydrous ethanol) to the column, and run at 12000 × g for 1 min.

[0046] (6) Repeat step (5).

[0047] (7) Discard the filtrate, put the column back into the collection tube, 12000×g 2 min.

[0048] (8) Place the column in a 1.5 mL centrifuge tube, add 25 μL of sterile water to the center of the column membrane, let stand at room temperature for 2 min, and then centrifuge at 12000 × g for 1 min.

[0049] (9) Discard the column and store the collected DNA in a -20°C freezer.

[0050] 3) Ligation of PCR products

[0051] Take 1 μL of the purified product and determine the concentration of the recovered PCR product using a spectrophotometer. Follow the instructions of the vector kit (pMD™ 18-T Vector Cloning Kit):

[0052] Ligation system: 0.8 μL vector, 4.2 μL PCR product, 5.0 μL Solution I, for ligation.

[0053] Connection conditions: 16℃, connection for 16 hours.

[0054] 4) Transformation into competent cells

[0055] (1) Take 100 μL of competent cells (DH 5α) from the -80℃ freezer and place them on ice to thaw. Add 10 μL of ligation product, mix gently, and let stand on ice for 30 min.

[0056] (2) Heat shock in a 37°C water bath for 50 s, then quickly place on ice to cool for 5 min.

[0057] (3) Add 900 μL of antibiotic-free LB liquid medium and incubate at 37°C with shaking at 200 rpm for 1 h.

[0058] (4) After centrifuging at 4000 rpm for 3 min, discard 900 μL of the upper culture medium, resuspend the bacterial cells, take 100 μL of bacterial solution and spread it on LB plates containing antibiotics, and incubate upside down in a 37 ℃ constant temperature incubator for 12 h.

[0059] 5) Screening and identification of positive clones

[0060] Single colonies were picked from the screening plates and incubated in 900 μL of LB broth containing antibiotics for 4–6 h. Positive clones were then verified using colony PCR. The specific reaction procedure is as follows:

[0061]

[0062] Reaction program: 95℃ for 3 min, (95℃ for 15 s, 55℃ for 30 s, 72℃ for 1 min) × 30, 72℃ for 7 min.

[0063] After the PCR reaction, 5 μL of the PCR product was subjected to 1% agarose gel electrophoresis for detection. 200 μL of the positive clone with the correct insert size was selected and sent to Guangzhou Tianyi Huiyuan Company for sequencing. BLAST homology comparison confirmed that the COI gene sequencing results were completely identical to the mitochondrial genome sequencing results, verifying the authenticity of the sequence.

[0064] 4. Bioinformatics Analysis

[0065] Nucleic acid sequences of related homologous proteins were searched using the NCBI database. The sample test sequences were named Hyotissa SP; Crassostrea gigas, KJ855245.1; Crassostrea sikamea, FJ841966.1; Crassostrea hongkongensis, FJ841963.1; Crassostrea ariakensis, EU672835.1; Crassostreairedalei, FJ841967.1; Saccostrea mordax, KP769562.1; Saccostrea kegaki, KT936587.1; Saccostrea cucullata, MF198445.1; Saccostrea malabonensis, ON649706.1; Neopycnodonte cochlear, AB076939.1; Pycnodonte taniguchii, AB076916.1; Hyotissa imbricata, AB076917.1; Hyotissa hyotis, GQ166583.1; Cellana orientalis, NC_063771.1; Clithon squarrosum, NC_062613.1; Lunellacorreensis, MT185943.1; Lineus viridis, FJ839919.1; Nipponnemertes punctatula, KC710980.1; Viviparus chui, KY679829.1; Cultellus attenuatus, MW653805.1. A phylogenetic tree was constructed using MEGA 6.0 software, and the neighborhood-joining method was used for analysis. The results showed that this sequence belongs to the same clade as that of *Hyotissa hyotis*, therefore, this sequence belongs to *Hyotissa hyotis*. Figure 2 B).

Claims

1. A lingulata oyster DNA barcode identification primer, characterized in that, The nested peripheral primer comprises HSCOI OU F: 5'-ATGCGTTGGGAGTTTGTGGTA-3' and HSCOI OU R: 5'-TCGAAAGAACATAATGAAAGTGGC-3'; and the nested internal primer comprises HSCOI IN F: 5'-CTATAAACGGCACTGGATGGA-3' and HSCOI IN R: 5'-AAAGACACGTCAACAGAACCC-3'.

2. A Crassostrea lingulata identification kit, characterized in that, The DNA barcode identification primer of the tongue bone oyster according to claim 1 is contained.

3. A method for identifying Crassostrea staminea, characterized by, The method comprises the following steps: The mitochondrial genome of the sample to be detected is extracted, the nested peripheral primer in claim 1 is used for first-time PCR amplification to obtain a first-time amplification product, the first-time amplification product is used as a template, the nested internal primer in claim 1 is used for second-time amplification to obtain a second-time amplification product, if the amplification products can be obtained in both times and the second-time amplification product is 1012 bp in size, the sample is the tongue bone oyster, otherwise, the sample is not the tongue bone oyster.

4. The method of claim 3, wherein, The PCR amplification system of the first-time PCR amplification is as follows: 1 ul of template, 9.5 ul of ultrapure water, 12.5 ul of 2x Taq PCR Master Mix, 2 ul of 1 mmol / L HSCOI OU F and 2 ul of 1 mmol / L HSCOI OU R.

5. The method of claim 3, wherein, The PCR amplification procedure of the first-time PCR amplification is as follows: 94 DEG C for 5 min; 94 DEG C for 30 s, 51 DEG C for 50 s, 72 DEG C for 1.2 min, 30 cycles; 72 DEG C for 10 min.

6. The method of claim 4, wherein, The PCR amplification system of the second-time PCR amplification is as follows: 1 ul of template, 9.5 ul of ultrapure water, 12.5 ul of 2x Taq PCR Master Mix, 2 ul of 1 mmol / L HSCOI IN F and 2 ul of 1 mmol / L HSCOI IN R.

7. The method of claim 3, wherein the step of identifying is characterized by, The PCR amplification procedure of the second-time PCR amplification is as follows: 94 DEG C for 5 min; 94 DEG C for 30 s, 51 DEG C for 50 s, 72 DEG C for 1.2 min, 30 cycles; 72 DEG C for 10 min.

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