Method for rapidly identifying crassostrea rivularis and crassostrea gigas based on losslessness and application of method

Abstract

The invention discloses a method for rapidly identifying crassostrea rivularis and crassostrea gigas based on lossless identification and application of the method, and belongs to the technical field of shellfish ecological restoration. The method comprises the following steps: measuring the content of the magnesium element in the ostrea rivularis shell powder and the crassostrea gigas shell powder, and identifying according to the difference of the content of the magnesium element. The method is different from a traditional molecular identification technology depending on the soft part of the oyster, and nondestructive, rapid and accurate identification of the ostrea rivularis and the crassostrea gigas can be realized by measuring the content of the magnesium element in the oyster shell. According to the method provided by the invention, even in an oyster reef complex sea area and a severely degraded area, the determination of reef building species can be realized by obtaining the oyster shell sample, and the development of oyster reef ecological restoration and conservation related work is greatly promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of shellfish ecological restoration, and particularly to a method for non-destructively and rapidly identifying Crassostrea ariakensis and Crassostrea gigas and its application. Background Art

[0002] As a typical representative of shellfish reefs, oyster reefs are known as "ecosystem engineers" and have many ecological service functions such as carbon sequestration, water purification, nitrogen removal, and increasing biodiversity. However, under the combined effects of human activities and global climate change, oyster reef resources have seriously degraded worldwide (about 85%). To reverse this trend, oyster reef ecosystem restoration and conservation work has been successively carried out in many places around the world. Identifying the oyster species that build reefs is the basic information for judging the health status of the existing oyster reef ecosystem and is also a prerequisite for large-scale ecological restoration projects.

[0003] Crassostrea ariakensis and Crassostrea gigas are the main reef-building species coexisting in estuaries and coastal areas in northern China. In the past, for the species identification of oysters (CN105734134A), the molecular identification method of extracting DNA from the soft parts was usually used. However, in the actual sampling process of oyster reefs, due to the limitations of the complex marine environment, it is often impossible to obtain live oysters, so the species cannot be accurately identified. Given that oyster reef resources have seriously degraded, the species identification method at the cost of live dissection of oysters has also damaged the local oyster resources to a certain extent, which goes against the original intention of oyster reef ecological restoration and ecological conservation. Therefore, there is an urgent need to find an alternative method that can identify oyster species non-destructively and rapidly. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for non-destructively and rapidly identifying Crassostrea ariakensis and Crassostrea gigas and its application to solve the problems existing in the above-mentioned prior art. The method provided by the present invention can identify Crassostrea ariakensis and Crassostrea gigas non-destructively, rapidly and accurately.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] Technical Solution 1: It includes the steps of measuring the magnesium element content in the shell powder of Crassostrea ariakensis and the shell powder of Crassostrea gigas, and identifying according to the difference in the magnesium element content.

[0007] Further, the identification according to the difference in the magnesium element content includes: when the magnesium element content is 1200-1400 mg / kg, it is identified as Crassostrea ariakensis; when the magnesium element content is 2000-2300 mg / kg, it is identified as Crassostrea gigas.

[0008] Further, the steps specifically include: digesting the Crassostrea ariakensis shell powder and the Crassostrea gigas shell powder respectively; measuring the magnesium element content; and identifying based on the magnesium element content.

[0009] Further, the preparation of the Crassostrea ariakensis shell powder and the Crassostrea gigas shell powder includes: cleaning and grinding the Crassostrea ariakensis shells and the Crassostrea gigas shells.

[0010] Further, the cleaning is: soaking with a 5%-10% sodium hypochlorite solution; and passing through a 2000-mesh sieve after grinding.

[0011] Further, the digestion is carried out using high-purity nitric acid.

[0012] Further, the conditions for the digestion are: heating at 150°C for 2 hours, and continuing to digest for 2 hours after raising the temperature to 180°C.

[0013] Further, the measurement of the magnesium element content is carried out using an inductively coupled plasma optical emission spectrometer.

[0014] Further, the identification based on the difference in the magnesium element content includes: identifying as Crassostrea ariakensis when the magnesium element content is 1200-1400 mg / kg; and identifying as Crassostrea gigas when the magnesium element content is 2000-2300 mg / kg.

[0015] Technical solution two: Application of the method in promoting the ecological restoration and conservation of oyster reefs.

[0016] The present invention discloses the following technical effects:

[0017] The present invention realizes species identification by collecting oyster shell samples, washing and grinding them, and then measuring the content of magnesium (Mg) element. By using the significant difference in Mg content between Crassostrea rivularis (1200 - 1400 mg / kg) and Crassostrea gigas (2000 - 2300 mg / kg), species identification is achieved. Compared with the traditional molecular identification technology that relies on DNA extraction from the soft parts, the present invention does not require destroying live oysters, thus solving the problems of difficult acquisition of samples in complex sea areas and ecological damage (traditional reef-building species identification relies on the molecular identification method of DNA extraction from the soft parts of live oysters, but in the actual process of collecting oyster reef samples, it is often impossible to obtain live oysters due to the limitations of the complex marine environment; on the other hand, for sea areas where the oyster reef ecosystem has been severely damaged, the species identification method at the cost of live dissection of oysters also damages the local oyster resources to a certain extent, contrary to the original intention of oyster reef ecological restoration and conservation). At the same time, the operation process is simplified, and the transportation and preservation costs are reduced (the present invention uses oyster shells as the identification materials for Crassostrea rivularis and Crassostrea gigas species, avoiding the limitations of low-temperature (-20°C) preservation and transportation required in the preservation and transportation of live oyster samples by the traditional method. The species identification of Crassostrea rivularis and Crassostrea gigas can also be achieved through normal-temperature preservation and long-distance transportation). The identification accuracy rate reaches 100%. The present invention can achieve non-destructive, rapid, and accurate identification of Crassostrea rivularis and Crassostrea gigas species by measuring the Mg element content in oyster shells, ensuring that even in complex sea areas and severely degraded areas of oyster reefs, reef-building species identification can be achieved by obtaining oyster shell samples, providing effective basic information for the restoration and conservation of oyster reef ecosystems. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 For the determination results of magnesium element content in Crassostrea rivularis and Crassostrea gigas, *** represents a significant difference between groups, p < 0.001;

[0020] Figure 2 For the phylogenetic tree showing the alignment of the DNA of the sequenced samples with the COI sequences of the two oysters in the NCBI database, the DNA sequences of the samples are clustered using the NJ phylogenetic tree and bootstrapped 1000 times. In the figure, AR is the abbreviation of Crassostrea rivularis, and GI is the abbreviation of Crassostrea gigas. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0025] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0026] Example 1 Collection of Oyster Shells

[0027] Thirty oyster shells were collected from the artificial oyster reef in Dongying Hekou District, and tissue samples were reserved for storage at -20 °C for future use to verify the accuracy of this method using traditional molecular identification techniques.

[0028] 1. Cleaning and Crushing of Oyster Shells

[0029] Each oyster shell was soaked in 5%-10% sodium hypochlorite to thoroughly remove the residual tissue and cutin layer. After soaking and cleaning twice until it became clear and bright, the cleaned shells were polished with 500-mesh, 1000-mesh, 2000-mesh, and 4000-mesh sandpapers until the cutin layer on the surface was completely removed. After drying in the sun, each individual oyster shell was ground into powder with a mortar. The oyster shell powder was filtered for impurities with a 2000-mesh sieve, and then waited to be measured for the Mg element content on the machine.

[0030] 2. Determination of Mg element in oyster shell powder

[0031] Accurately weigh the sample to be measured into a 50 mL high-foot beaker, moisten it with a small amount of ultrapure water, add 4 mL of high-purity nitric acid, cover the lid, and digest it on an electric heating plate at 150 °C for 2 h, then raise the temperature to 180 °C and continue to digest for 2 h until complete digestion. After cooling, open the lid and continue to heat until nearly dry. Add 1 mL of HNO3 solution of equal volume, and extract it slightly heated (temperature is 50 °C). Finally, transfer all the digestion solution to a 15 mL centrifuge tube, wash the beaker 4 times with deionized water, combine the washing solutions, and make up to the scale line with deionized water, shake well and set aside for use on the machine (the machine manufacturer is PerkinElmer, Inc. and the model is Optima 7300DV). Do a reagent blank experiment in the same way.

[0032] 3. Data processing of magnesium (Mg) element in oyster shell powder

[0033] Process the data obtained from the machine. Convert the Mg element content of each individual oyster shell powder into mg / kg for comparison. The results are as Figure 1 shown. Among the 30 test samples, the Mg element content of 4 portions of oyster shell powder is 1250 - 1386 mg / kg, which is determined to be Crassostrea rivularis. The Mg element content of 26 portions of oyster shell powder is 2053 - 2214 mg / kg, which is determined to be Crassostrea gigas. The determination principle is: the Mg element content of Crassostrea rivularis shell is 1200 - 1400 mg / kg, and the Mg element content of Crassostrea gigas shell is 2000 - 2300 mg / kg.

[0034] 4. Verify the accuracy of this method by applying the molecular identification method

[0035] Verify the accuracy rate of the above-mentioned identification method provided by the present invention according to the molecular identification method disclosed in Wang et al., (2008) (the detailed information of the article is H. Wang and X. Guo, 2008. Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR. Journal of Shellfish Research, Vol. 27, No. 3, pp: 489-493). According to the disclosure of this literature, artificially synthesize the COI primers, extract DNA and amplify PCR products from the soft parts of the above-mentioned 30 identified oysters, sequence the PCR products by Sanger's dideoxy chain termination method, and use Bioedit and MEGA software for sequence alignment and phylogenetic tree analysis. The results are as Figure 2 shown (using the NJ phylogenetic tree to cluster the DNA sequences of the samples, with 1000 bootstrap replicates). It can be seen that the 4 samples inferred as Crassostrea rivularis by the identification method provided by the present invention are clustered into one branch with the COI sequence of Crassostrea rivularis in the NCBI database (NCBI number: KJ855253.1), and the average genetic distance is 0.019 - 0.004; in addition, the 26 samples inferred as Crassostrea gigas by the identification method provided by the present invention are clustered into one branch with the COI sequence of Crassostrea gigas in the database (NCBI number: OY970755.1), and the average genetic distance is 0.004 - 0.067. The intraspecific genetic distances of both are much smaller than the interspecific genetic distance (0.182 - 0.230). Therefore, the identification method provided by the present invention can accurately identify Crassostrea rivularis and Crassostrea gigas according to the Mg element results of individual oyster shell powder, and the accuracy rate of species identification is 100%.

[0036] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for identifying Crassostrea rivularis and Crassostrea gigas, characterized in that It includes the steps of measuring the magnesium element content in the Crassostrea rivularis shell powder and the Crassostrea gigas shell powder, and identifying based on the difference in the magnesium element content.

2. The method according to claim 1, wherein Identifying based on the difference in the magnesium element content includes: identifying as Crassostrea rivularis when the magnesium element content is 1200 - 1400 mg / kg; identifying as Crassostrea gigas when the magnesium element content is 2000 - 2300 mg / kg.

3. The method according to claim 1, wherein The specific steps include: digesting the Crassostrea rivularis shell powder and the Crassostrea gigas shell powder respectively; measuring the magnesium element content; and identifying based on the magnesium element content.

4. The method according to claim 2, wherein The preparation of the Crassostrea rivularis shell powder and the Crassostrea gigas shell powder includes: cleaning and grinding the Crassostrea rivularis shell and the Crassostrea gigas shell.

5. The method according to claim 4, wherein The cleaning is: soaking with a 5% - 10% sodium hypochlorite solution; and passing through a 2000 - mesh sieve after grinding.

6. The method according to claim 3, wherein The digestion is carried out with high-purity nitric acid.

7. The method according to claim 3, characterized in that The conditions for the digestion are: heating at 150 °C for 2 hours, and continuing digestion for 2 hours after raising the temperature to 180 °C.

8. The method according to claim 3, wherein The measurement of the magnesium element content is carried out using an inductively coupled plasma optical emission spectrometer.

9. The method according to claim 3, wherein Identifying based on the difference in the magnesium element content includes: identifying as Crassostrea rivularis when the magnesium element content is 1200 - 1400 mg / kg; identifying as Crassostrea gigas when the magnesium element content is 2000 - 2300 mg / kg.

10. Application of the method according to any one of claims 1 - 9 in promoting the ecological restoration and conservation of oyster reefs.

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