CN112626238A - Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method - Google Patents
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Abstract
The invention discloses a characteristic sequence, a specificity identification primer and an identification method for identifying a crassostrea sikamea, wherein the characteristic sequence is positioned in a mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of the crassostrea sikamea, and is a nucleotide sequence shown as SEQ ID NO. 1; the sequence of the specific identification primer is as follows: the upstream primer MNR-F: 5'-CTGTAAGTATATTTGTCTTCCA-3', respectively; the downstream specific primer MNR-S-R: 5'-AGGCTTTCACTCCACTTACT-3' are provided. The invention can effectively distinguish the oyster from other four oyster species of the genus oyster, the determined characteristic sequence length of the oyster is 660 +/-5 bp, and whether the oyster is the oyster can be judged according to the existence and the size of the specific strip. In the field of the protection of the genetic resources of the crassostrea sikamea and the breeding of the crassostrea sikamea, species identification can be carried out on parent crassostrea sikamea before breeding of the crassostrea sikamea to ensure that the bred crassostrea sikamea is pure and is not influenced by other species possibly hybridized with the bred crassostressostressostressostrea sika.
Description
Technical Field
The invention relates to the fields of molecular biology and aquatic germplasm resource protection and application, in particular to a characteristic sequence, a specific identification primer and an identification method for identifying Crassostrea sikamea.
Background
Oysters, commonly known as oysters, oyster yellow and the like, are distributed in the world, have more than 600 million tons of annual output in the world, and are important seawater cultured shellfishes. Oyster resources are abundant in China, breeding varieties are various, breeding varieties in Liaoning province, Shandong province and other provinces in the North are Crassostrea gigas, Fujian province is Fujian oyster (C.angulata), Guangdong province is hong Kong oyster (C.hongkongensis) and Yuanjiang oyster (C.ariakensis). The area of the sea area in Zhejiang province is wide, important culture seaports such as hong Kong, Loqing bay and Sanmenbay exist along the sea, tidal flat shellfish such as oyster are one of main culture types, and the yield of the oyster in Zhejiang province in 2018 reaches 22 million tons (Fishery agency of Ministry of agriculture, 2019). In addition, the oyster fries in the region are mainly natural seedlings, and the recent identification results of the inventor on the oyster species in three main culture gulfs in Zhejiang province show that the Crassostrea gigas and the Crassostrea gigas are mainly distributed along the Zhejiang coast, wherein the Crassostrea gigas are dominant species and are supposed to be indigenous species.
The crassostrea sikamea is named as the first discovery in japan, which is smaller in size and deeper in the left shell than crassostrea gigas and pacific oysters. It was introduced from japan to the west coast of the united states as crassostrea gigas in the forties of the last century, and was artificially bred and cultured. The mouthfeel of the crassostrea sikamea is generally considered to be superior to that of other crassostrea sikamea species, and the crassostrea sikamea gradually becomes a high-end crassostrea sikamea species. In addition, since the crassostrea sikamea is naturally distributed in japan, korea and south coast of south china Jiangsu, population genetics studies indicate that the genetic diversity of crassostrea sikamea in the Zhejiang coast is significantly higher than that of the populations in japan and korea, which may be the origin of crassostrea sikamea.
Because the shell shape of the oyster is changeable, species identification of the oyster based on the shell shape is difficult, and the phenomenon that the oysters bear and other species are distributed in the same domain exists in a plurality of sea areas in Japan and south China, and in addition, the oysters can be fertilized with Fujian oysters, hong Kong oysters and Crassostrea gigas in a single direction to generate offspring under the condition of artificial propagation. The training of the crassostrea sikamea population from the united states is worth deep thinking, after the crassostrea sikamea is introduced into the united states, species can be judged only through a shell type due to lack of a species molecular identification method, so that a gene bank of the crassostrea sikamea is polluted by pacific oysters due to intentional or unintentional hybridization, genetic diversity of the crassostrea sikamea is reduced, effective population is reduced, and it is extremely important to remind researchers to accurately perform species identification before other works are started.
In the past decade, researchers have established a series of molecular identification methods for oyster species, and target genes include endogenous transcribed spacer 1 (ITS 1), and mitochondrial genes such as cytochrome oxidase i (coi) and common genetic markers of 16S. The identification method includes a method for distinguishing between the crassostrea sikamea and the pacific oyster using ITS1 gene based on Hae III and Hind III restriction enzymes. The method for distinguishing seven oysters in the coastal region of the south of China by using PCR-RFLP analysis, the method for distinguishing five oyster species of oyster genus by using multiple PCR based on COI gene, and the high-resolution melting analysis method based on COI and independent of species specificity PCR or fragment length polymorphism. For example, the 200710030295 patent application discloses a PCR-RFLP identification method for seven crassostrea oysters in the coastal region of south China, which mainly aims at various crassostrea species including the crassostrea gigas, wherein the PCR product needs to be subjected to double enzyme digestion, and the number of bands after enzyme digestion is more and is 1-5, so that the identification is not easy to develop and has errors. 201210189381 patent application discloses a method for identifying oyster closely related species, which is aimed at various oyster species including crassostrea gigas, by PCR amplification and utilizing the Tm value difference of the amplification product in a high resolution melting curve (HRM) to distinguish oyster species. The multiplex PCR method (Wang et al 2008) aims at various crassostrea species, the species of the crassostrea are judged according to the length of a product by a plurality of specific PCR primers of a coding region of a COI gene, but in practice, as many as 6 primers need to be added, and the COI is used as a coding gene, so that the difference among the species is limited, and non-specific amplification can be generated to cause the non-singleness of a product band, thereby failing to identify. 201610165034 the method for molecular discrimination between Ostrea rivularis and Ostrea ursinus disclosed in the patent application can be used for discriminating Ostrea rivularis and Ostrea ursinus only, and has a limited application range. 201711041921 patent application discloses a method for identifying crassostrea sikamea based on DNA specific fragments, which can specifically identify crassostrea sikamea from widely distributed crassostrea sikamea, and the target band of PCR product in the method is short, only about 100bp, and is not easily distinguished from non-target bands such as primer dimer. In conclusion, the method for identifying the important species of the oyster breeding, namely the crassostrea sikamea, with stronger pertinence and simplicity needs to be established urgently.
Disclosure of Invention
The invention aims to solve the technical problem that a characteristic sequence, a specificity identification primer and an identification method for identifying the oysters with the sika oyster characteristics are provided aiming at a mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of the oysters, and can play an important role in the germplasm resource protection, artificial propagation and related biological research of the sika oysters.
The inventor discovers that the sequence lengths of mitochondrial non-coding regions of the oyster oysters of the oyster genera have certain difference between glycine tRNA synthetase and proline tRNA synthetase by analyzing the existing mitochondrial genome sequences of the oyster oysters of the genera oyster in a database, and the sequence lengths of the oyster oysters of the genera oyster are between 700-815bp, so that the sequence polymorphism is high. The non-coding region is amplified by designing universal primers on two coding genes at two sides of a target region, and the adopted upstream primer MNR-F is as follows: 5'-CTGTAAGTATATTTGTCTTCCA-3', the downstream primer MNR-R is: 5'-GACGTTGTAAGCGTCATGTAAT-3', the sequence length of the region was found to be conserved between individuals of the same species. Through detection, the lengths of the sequences corresponding to different species are respectively as follows: the oyster comprises 749bp of the sika oyster, 739bp of the Fujian oyster, 737bp of the crassostrea gigas, 812bp of the crassostrea rivularis and 704bp of the hong Kong oyster. But the differences based on sequence length were not sufficient to distinguish the oysters. The inventors of the present invention analyzed a larger number of crassostrea sikamea sequences in the region by amplification, found that the crassostrea sikamea sequences have a large difference from other species around 660bp downstream of the upstream primer sequence, and designed downstream specific primers based on the region. Therefore, the invention provides a primer and a method for identifying the characteristic sequence and the specificity of the crassostrea sikamea.
The technical scheme adopted by the invention for solving the technical problems is as follows: identifying the characteristic sequence of the oysters, wherein the characteristic sequence is located in a mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of the oysters, and is a nucleotide sequence shown in SEQ ID NO. 1.
The specific identification primer for identifying the crassostrea sikamea has a sequence as follows:
the upstream primer MNR-F: 5'-CTGTAAGTATATTTGTCTTCCA-3', respectively;
the downstream specific primer MNR-S-R: 5'-AGGCTTTCACTCCACTTACT-3' are provided.
The identification method for identifying the crassostrea sikamea specifically identifies the crassostrea sikamea based on a mitochondria non-coding region and comprises the following steps:
(1) dissecting the collected crassostrea sikamea, sampling the adductor muscle tissue, quickly freezing by liquid nitrogen, and storing in a low-temperature refrigerator;
(2) extracting genomic DNA of the oyster by using a kit to obtain a DNA stock solution, and diluting the DNA stock solution to 1-10 ng/mu L after the quality inspection is qualified to be used as a PCR template for later use;
(3) designing an upstream primer MNR-F and a downstream specific primer MNR-S-R as specific identification primers for species identification, wherein the upstream primer MNR-F is 5'-CTGTAAGTATATTTGTCTTCCA-3', and the downstream specific primer MNR-S-R is 5'-AGGCTTTCACTCCACTTACT-3';
(4) taking the genomic DNA of the oyster extracted in the step (2) as a template, and performing PCR amplification reaction by using a designed specific identification primer to obtain a PCR product of a target fragment;
(5) and (3) carrying out 1.2% agarose gel electrophoresis on the PCR product under the conditions of 220V and 200mA to obtain a product strip with the size of 660 +/-5 bp, namely the characteristic sequence SEQ ID NO.1 of the crassostrea sikamea.
Preferably, the reaction procedure of the PCR amplification reaction in step (4) is: pre-denaturation at 94 ℃ for 3 min; another 30 cycles were performed: 94 ℃ for 30 seconds, 50 ℃ for 30 seconds, 72 ℃ for 50 seconds; final extension at 72 ℃ for 10 min.
Compared with the prior art, the invention has the following advantages: the invention determines the characteristic sequence of the oyster based on the characteristics of high polymorphism and large difference between species of mitochondrial non-coding region sequences between glycine tRNA synthetase and proline tRNA synthetase of oysters of different species, develops a specificity identification primer and an identification method for identifying the oyster, can effectively distinguish the oyster from other four oyster species of giant oyster, determines the characteristic sequence length of the oyster to be 660 +/-5 bp, does not have specificity strips of other four oyster species of giant oyster, and can judge whether the oyster is the oyster or not according to the existence and the size of the specificity strips. In the field of the protection of the genetic resources of the crassostrea sikamea and the breeding of the crassostrea sikamea, species identification can be carried out on parent crassostrea sikamea before breeding of the crassostrea sikamea to ensure that the bred crassostrea sikamea is pure and is not influenced by other species possibly hybridized with the bred crassostressostressostressostrea sika. The identification method has the advantages that the method does not depend on expensive instruments, does not need sequencing, does not need enzyme digestion on PCR products, is simple, convenient and clear to operate, can quickly finish the identification of the crassostrea sikamea with high specificity, has consistent results with sequencing verification results, and does not identify individuals by errors.
Drawings
FIG. 1 shows the amplification results of five specific primers for oyster of genus oyster in example 1;
FIG. 2 shows the sequence alignment results of the universal upstream primers for five species of oyster in the genus oyster in example 1;
FIG. 3 shows the alignment results of the downstream specific primer sequences of five species of Crassostrea oysters in example 1;
FIG. 4 shows the validation results of oyster specific primers in example 2 at four spots.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1:
a) sample collection
Collecting crassostrea gigas cultured in Qingdao sea area, crassostrea gigas cultured in Fujian sea area, crassostrea gigas cultured in Guangdong sea area, crassostrea hongkongensis and crassostrea gigas cultured in Nippon sea area of Zhejiang, randomly sampling 30 oysters in each place, placing the adductor muscle into a freezing tube after dissecting the oysters, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃;
b) extraction of DNA
1) Taking about 20mg of tissues of each oyster individual in a 1.5mL sterilized centrifugal tube, and performing DNA extraction by using a DNA kit to obtain a DNA stock solution;
2) carrying out 1.2% agarose gel electrophoresis on the extracted DNA stock solution to detect the integrity of the DNA, and detecting the concentration and the purity of the DNA by using Nanodrop;
3) diluting the extracted DNA stock solution to 1-10 ng/mu L with sterilized water for subsequent PCR amplification;
c) species molecular identification
Performing PCR by using MNR and COI universal primers to amplify sequences, sequencing PCR products, and performing sequence comparison on sequencing results in an NCBI database to verify the correctness of species of the crassostrea gigas, the Fujian oyster, the crassostrea rivularis, the hongkongensis oyster and the crassostrea sikamea obtained by sampling;
d) specific primer PCR amplification verification
Randomly selecting the five oysters, and carrying out specific primer verification on 5 oysters respectively, wherein an upstream primer MNR-F is 5'-CTGTAAGTATATTTGTCTTCCA-3', a downstream specific primer MNR-S-R is 5'-AGGCTTTCACTCCACTTACT-3', a reaction system is shown in the following table 1, and the reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; another 30 cycles were performed: 94 ℃ for 30 seconds, 50 ℃ for 30 seconds, 72 ℃ for 50 seconds; final extension at 72 ℃ for 10 min. The amplification results of the five oyster specific primers of oyster genus of oyster species of example 1 are shown in FIG. 1. In FIG. 1, the wells 1-5 are Crassostrea sikamea, 7-11 are Crassostrea gigas, 12-16 are Crassostrea gigas, 18-21 are Crassostrea rivularis, 22-25 are Crassostrea hongkongensis, and 6 and 17 are on the molecular scale (DL1000, the maximum band is 1000bp, and the second band is 700 bp). FIG. 2 shows the sequence alignment results of the universal upstream primers for five species of oyster in the genus oyster in example 1; FIG. 3 shows the alignment results of the downstream specific primer sequences of five species of oyster species of the genus Crassostrea in example 1. The result shows that only the oysters can amplify specific bands with the length of 664bp, the characteristic sequence corresponding to the specific bands is shown as SEQ ID NO.1 (the types are nucleic acids, the strand type is single-chain, the topological structure is linear, and the molecular type is DNA), and other species have no specific bands.
TABLE 1 PCR reaction System
Example 2:
a) sample collection
Collecting intertidal oyster samples of three bays-Xiangshan harbor, Sanmenwan, Leqing bay and Fujian Zhangzhou in Zhejiang coastal sea, randomly taking 30 oysters at each place, quickly freezing the adductor muscle in liquid nitrogen after oyster dissection, and storing in a refrigerator at-80 ℃ for subsequent verification;
b) DNA extraction and oyster species molecular identification
Extracting DNA by using a DNA kit according to an instruction, performing MNR and COI sequence amplification after detecting the purity and concentration of the DNA, determining oyster species mainly including two species of the crassostrea sikamea and the crassostrea fujianensis by NCBI Blast after sequencing a PCR product (only the crassostrea sikaensis is taken in the gulf of le Qing);
c) specific primer PCR amplification verification
3 oysters in the four places are randomly selected from each species for specific primer verification, the upstream primer MNR-F is 5'-CTGTAAGTATATTTGTCTTCCA-3', the downstream specific primer MNR-S-R is 5'-AGGCTTTCACTCCACTTACT-3', and the reaction system and the PCR program are as in example 1. FIG. 4 shows the validation results of oyster specific primers in example 2 at four spots. In FIG. 4, the glue holes 1-3 are Crassostrea gigas collected from hong kong, and 4-6 are Crassostrea gigas collected from Fujian province; 7-9 is Concha Ostreae collected from Sanmenwan, and 10-12 is Concha Ostreae of Fujian province; 13-15 of the oyster collected in Zhangzhou, and 16-18 of the oyster collected in Fujian; 20-22 were crassostrea sikamea collected in the gulf of le qing, and 19 was a molecular scale (DL2000, maximum band of 2000bp, brightest band of 750 bp).
The results show that primers of oyster samples collected from three places along the coast of Zhejiang and Zhangzhou of Fujian prove that the oyster has specific strips, the length of the oyster is about 660bp, and other oysters have no specific strips.
Sequence listing
<110> Ninghai Marine organism species research institute of Wanli institute of Zhejiang, and Wanli institute of Zhejiang
<120> identification of characteristic sequence, specificity identification primer and identification method of crassostrea sikamea
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 664
<212> DNA
<213> Crassostrea sikamea characteristic sequence located in mitochondrial noncoding region between glycine tRNA synthetase and proline tRNA synthetase of Crassostrea sikamea (SEQ ID NO. 1)
<400>1
CTGTAAGTATATTTGTCTTCCAAACAAAAGGTTTTCGTAAATGAAAAAAGCGTATGATTATATTAAAGTTGGAAGTTTAATTTTTAAGCAATCACAGCGGGTAAAAAATACTTAAAGTTAGCTTGTTACTGGGATTGCTTTAATTTTGATATTTTTAATAATCTATTTTTAAGCCTCTGTGACATGACGTAAATTTTTTGAATTTTCTTAGAATTAAGTTATTTTTACCTTATGTGTTAACCGGTTGCTTTGTGAGTTGAAATACAGCTTAAGTAAAAAAAATAAAATATCTTTTCCCTATGTAATAGTAGAATTTTAACATCTTGACTTATAGCGATTTGTGCTTAGTTTGTGGTAATATGGGGGGTAAAACTAAAGTTTGATACCGCATGAATTGCTTATAAGCAGGCTTAAAATGAGTTTGATTTATTGGATGGCAGAATTAAAAAATGTTGAAGTGAAGTTTTTTAGAAGTAGGAAGTATTAAAAATTAATTCTAGTGGTTGTATGAGTTGTAAGATTGGGTATATAATGGTAAAGGTTTAAAAGTGATAAAAGGTAAAAGGTGGGTATAATCTGTTTTGGTATAAAAATACATAGTCTTGGATCATGGGAAACTATAAAGGTGTAGTTATAAGTTTTAAAGTAAGTGGAGTGAAAGCCT
Claims (4)
1. The characteristic sequence of the oyster is identified, the characteristic sequence is located in a mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of the oyster, and the characteristic sequence is a nucleotide sequence shown in SEQ ID NO. 1.
2. The specificity identification primer for identifying the crassostrea sikamea is characterized by comprising the following sequences:
the upstream primer MNR-F: 5'-CTGTAAGTATATTTGTCTTCCA-3', respectively;
the downstream specific primer MNR-S-R: 5'-AGGCTTTCACTCCACTTACT-3' are provided.
3. The identification method for identifying the crassostrea sikamea is characterized in that the specific identification is carried out on the crassostrea sikamea based on a mitochondria non-coding region, and the identification method specifically comprises the following steps:
(1) dissecting the collected crassostrea sikamea, sampling the adductor muscle tissue, quickly freezing by liquid nitrogen, and storing in a low-temperature refrigerator;
(2) extracting genomic DNA of the oyster by using a kit to obtain a DNA stock solution, and diluting the DNA stock solution to 1-10 ng/mu L after the quality inspection is qualified to be used as a PCR template for later use;
(3) designing an upstream primer MNR-F and a downstream specific primer MNR-S-R as specific identification primers for species identification, wherein the upstream primer MNR-F is 5'-CTGTAAGTATATTTGTCTTCCA-3', and the downstream specific primer MNR-S-R is 5'-AGGCTTTCACTCCACTTACT-3';
(4) taking the genomic DNA of the oyster extracted in the step (2) as a template, and performing PCR amplification reaction by using a designed specific identification primer to obtain a PCR product of a target fragment;
(5) and (3) carrying out 1.2% agarose gel electrophoresis on the PCR product under the conditions of 220V and 200mA to obtain a product strip with the size of 660 +/-5 bp, namely the characteristic sequence SEQ ID NO.1 of the crassostrea sikamea.
4. The method for identifying oysters, according to claim 3, wherein the reaction sequence of the PCR amplification reaction in step (4) is: pre-denaturation at 94 ℃ for 3 min; another 30 cycles were performed: 94 ℃ for 30 seconds, 50 ℃ for 30 seconds, 72 ℃ for 50 seconds; final extension at 72 ℃ for 10 min.
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| CN117551791A (en) * | 2024-01-11 | 2024-02-13 | 中国科学院海洋研究所 | Primer combination, application and identification method and kit for identifying Chinese common oyster by specific multiplex PCR |
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