A molecular marker, primer group and application for identifying gender of philippine clams

Abstract

The application discloses a molecular marker, a primer group and application for identifying gender of Manila clam, belongs to the technical field of shellfish gender identification, the sequence of the molecular marker is shown as SEQ ID NO.1, and the sequences of the primer group for amplifying the molecular marker are shown as SEQ ID NO.5 and SEQ ID NO.6; and a gender identification method of Manila clam is also developed, and the method comprises the following steps: extracting DNA of Manila clam to be tested; performing PCR amplification on the extracted DNA of the Manila clam to be tested by using the primer group to obtain a PCR product; performing electrophoresis analysis on the PCR product; if a band with a size of 600bp is observed, then the gender of the Manila clam to be tested is male; and if no band with a size of 600bp is observed, then the gender of the Manila clam to be tested is female. The application can quickly, accurately and sensitively identify the gender of the Manila clam.

Description

Technical Field

[0001] This invention belongs to the field of shellfish sex identification technology, specifically involving a molecular marker, primer set, and application for identifying the sex of Manila clams. Background Technology

[0002] Philippine clam (scientific name: Ruditapes philippinarum The Manila clam (Viburnum macrocephalum) is a type of shellfish belonging to the genus Viburnum in the family Veneridae. Commonly known as the Manila clam, it is characterized by rapid growth, a short cultivation cycle, strong adaptability, and long survival time out of water, making it suitable for high-density farming. It is one of the four major farmed shellfish in China. Studies have found that Manila clams exhibit sexual dimorphism and sex reversal, with females generally being larger and males generally smaller. Rapid sex differentiation is crucial for enhancing its economic value.

[0003] The Manila clam is a type of mollusks exhibiting double uniparental inheritance (DUI), with two types of mitochondrial DNA: F-type and M-type. F-type DNA carries maternal genetic information through the egg, while M-type DNA carries paternal genetic information through the sperm. Offspring sex is closely related to mitochondrial DNA inheritance. In female Manila clams, the mitochondrial DNA in the gonads and somatic tissues is homogeneous (F-type), while in males, the mitochondrial DNA is heterogeneous, primarily M-type mitochondrial DNA in the gonads.

[0004] Commonly used methods for sex determination include cytogenetic sex chromosome identification, microscopic examination, and sex-specific molecular markers. Cytogenetic methods determine sex using sex-specific sex chromosomes, but this method is not only time-consuming and labor-intensive, but also unfeasible for shellfish lacking sex chromosomes. Microscopic examination is currently a commonly used method for sex determination in shellfish, but this method is difficult for juvenile Manila clams because their reproductive cells in the gonads are not fully developed or have not developed at all, resulting in indistinct morphological characteristics and making accurate sex determination difficult. Sex-linked or sex-specific markers are widely used in biology and agriculture to determine the sex of individuals. Molecular sex determination is particularly important in species lacking easily distinguishable dimorphic phenotypes or individuals at developmental stages lacking secondary sexual characteristics. Currently, sex-specific DNA sequences or markers have been successfully developed for identifying a range of farmed fish and crustacean species, such as turbot. Scophthalmus maximus Random amplified polymorphic DNA (RAPD) of the swimming crab (Portunus trituberculatus) Portunus trituberculatus ) and Pacific bluefin tuna ( Thunnus orientalis Amplified fragment length polymorphism (AFLP) of ) and rock seabream ( Oplegnathus fasciatusThe study identified a simple sequence repeat (SSR) marker for sex determination in Manila clams. This discovery lays the foundation for exploring the molecular mechanisms of sex determination and differentiation in Manila clams, and is of great significance for their germplasm improvement and population management. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a molecular marker for sex identification of Manila clams and its application. Using the molecular marker provided by this invention, the sex of Manila clam samples can be accurately identified.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a molecular marker for sex identification of Manila clams, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A second aspect of the present invention provides a primer set for the specific detection of the molecular markers described above, the primer set comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6.

[0009] A third aspect of the present invention provides the application of the primer set described above in the preparation of a molecular diagnostic kit for identifying the sex of Manila clams.

[0010] A fourth aspect of the present invention provides a kit comprising the primer set, dNTPs, PCR stabilizer, Taq DNA polymerase and PCR buffer described above.

[0011] The fifth aspect of the present invention provides an application of the molecular markers or primer sets described above in sex identification of Manila clams, wherein the method is to identify sex by detecting the presence or absence of the molecular markers.

[0012] The sixth aspect of this invention provides an application of the aforementioned molecular markers or primer sets in the breeding or assisted breeding of Manila clams.

[0013] The seventh aspect of this invention provides a method for sex determination of Philippine clams, comprising the following steps:

[0014] DNA was extracted from the Manila clams to be tested;

[0015] The DNA extracted from the Manila clam was amplified by PCR using the primer set described above to obtain the PCR product.

[0016] The PCR products were analyzed by electrophoresis.

[0017] If the amplification product contains a 600bp fragment, the sex of the Manila clam being tested is male; if the amplification product does not contain a 600bp fragment, the sex of the Manila clam being tested is female.

[0018] Furthermore, each 20 μL PCR amplification reaction system includes: 1 μL~2 μL DNA template, 10 μL 2×PCR Mastermix, 0.1 μL~0.5 μL 10 μmol / L upstream primer, 0.1 μL~0.5 μL 10 μmol / L downstream primer, and deionized water to make up the difference.

[0019] Further, the reaction procedure for the PCR amplification is as follows: (1) pre-denaturation; (2) denaturation; annealing: 52.1℃~62.1℃, 45s; extension; a total of 25~30 cycles; (3) terminal extension.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention discloses for the first time a molecular marker for sex identification of Manila clams, the sequence of which is shown in SEQ ID NO.1. It also provides a primer set for detecting the molecular marker, a kit containing the primer set, and a method for sex identification of Manila clams. Compared with histological identification methods, the method provided by this invention has the advantages of not being limited by the degree of gonadal development, high accuracy, simple operation, easy result interpretation, short detection time, and low cost. This invention provides technical support for sex detection, identification, and breeding of Manila clams, lays the foundation for exploring the molecular mechanisms of sex determination and differentiation in Manila clams, and is of great significance for the improvement of Manila clam germplasm and population management. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 To expand M-type mitochondria NAD2 Electrophoresis results of primer sets amplified at three different cycle numbers; ×5, ×6, ×7, ×9, and ×10 represent five samples of Manila clams; M: DL 2000 molecular weight standard.

[0024] Figure 2 To expand F-type mitochondria NAD2Electrophoresis results of the primer set of the gene amplified in 67 samples of Manila clams; M: DL 2000 molecular weight standard; Figure B is a continuation of Figure A, and Figure C is a continuation of Figure B.

[0025] Figure 3 To expand M-type mitochondria NAD2 Electrophoresis results of primer sets amplified in 67 samples of Manila clams, M: DL 2000 molecular weight standard; Figure B is a continuation of Figure A, and Figure C is a continuation of Figure B. Detailed Implementation

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0027] Example 1: Obtaining molecular markers for sex identification of Manila clams

[0028] I. Experimental Materials

[0029] The experimental samples originated from Fujian Province. A total of 67 one-year-old sexually mature individuals were selected and temporarily housed in the aquatic animal breeding laboratory of Anhui Agricultural University for one week before being used in the experiment. The breeding water was a mixture of ecological sea salt and aerated water. During the temporary housing period, the temperature was maintained at 25℃±0.5℃, the dissolved oxygen content was stabilized at 7.9mg / L±0.2mg / L, the pH value was maintained at 7.0±0.1, and the salinity was maintained at 32‰±1‰. These physicochemical indicators were measured using a Hach SL1000 water quality analyzer. Chlorella was fed once daily as a food source, and the water was changed daily. Any individuals nearing or already dead were removed daily to maintain the stability of the breeding environment.

[0030] II. Experimental Instruments

[0031] The experimental instruments are shown in Table 1.

[0032] Table 1 Experimental Apparatus

[0033]

[0034] III. Experimental Methods

[0035] 1. Sampling

[0036] Healthy, vigorous Manila clams were selected as sampling subjects to ensure sample representativeness. During sampling, the adductor muscle of each individual was carefully severed to separate the two shells. The gonad was then punctured with the tip of a scalpel, and the gonadal tissue was gently separated from the surrounding tissue using a sterile dissecting needle and forceps, maintaining the tissue's integrity as much as possible. A suitable amount of gonadal tissue was collected with forceps and placed into a pre-prepared sterile, enzyme-free centrifuge tube. Sample information was recorded, and the tube was quickly placed in liquid nitrogen for rapid freezing to prevent ice crystals from damaging the cell structure and to preserve the sample's original state to the greatest extent possible. Simultaneously, the sex of the Manila clam was determined by microscopic observation of the reproductive cells within the gonad. The gonadal tissue sample in liquid nitrogen was then stored at -80°C.

[0037] 2. Extraction and detection of genomic DNA from gonadal samples

[0038] Genomic DNA was extracted from gonadal samples of Philippine clams using a marine animal tissue genomic DNA extraction kit.

[0039] First, cut 30mg of gonadal tissue into a centrifuge tube, add 200μL of buffer GA and 20μL of proteinase K to the centrifuge tube, centrifuge, and place in a 56℃ water bath. When the tissue is completely dissolved, centrifuge again to destroy the gonadal tissue cell structure and promote the release of DNA from the cells.

[0040] Then add 200 μL of buffer GB, invert thoroughly, heat at 70°C for 10 minutes, centrifuge after the solution becomes clear, add 200 μL of anhydrous ethanol, invert thoroughly to mix, and centrifuge again.

[0041] Next, add the solution and flocculent precipitate obtained in the previous step into the adsorption column and add 500 μL of buffer GD. After centrifugation, discard the waste liquid, add 600 μL of washing buffer, centrifuge again, and discard the waste liquid.

[0042] Finally, the purified DNA was released from the adsorption column by adding 50 μL of elution buffer, thus obtaining high-purity, high-quality genomic DNA.

[0043] The extracted genomic DNA was labeled and stored at -20°C to ensure its stability. The quality and concentration of the genomic DNA extracted from the gonadal tissue of the Manila clam were determined using an ultra-micro spectrophotometer, and the OD was accurately measured. 260 / OD 280 The ratio, obtained by measuring the OD of the sample's genomic DNA. 260 / OD 280 The quality of genomic DNA was determined by the value, and the known concentrations of the original DNA solution were diluted to 20 ng / μL to ensure that the DNA concentration met the requirements of later experiments.

[0044] 3. Establishment of a PCR-based sex determination system

[0045] Based on the mitochondrial protein coding gene sequences of Manila clams in the NCBI database, genes with large genetic distances between M-type and F-type sequences were first selected using MEGA 6.06 software. The selected M-type and F-type mitochondrial gene sequences were then subjected to differential alignment using MEGA 6.06 software, and fragments with significant differences were selected. These fragments were then compared with corresponding genome databases, excluding sequences with high homology to other coding sequences. Finally, M-type mitochondrial sequences were selected. NAD2 Genes, and using F-type mitochondria NAD2 Genes as a control, M-type mitochondria NAD2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, F-type mitochondria. NAD2 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0046] SEQ ID NO.1:

[0047] 。

[0048] SEQ ID NO.2:

[0049] .

[0050] According to M-type mitochondria NAD2 Genes and F-type mitochondria NAD2 The nucleotide sequence of the gene was obtained, and primers were designed using the PCR primer design software Primer Premier 5. Finally, the obtained primer sequences were given to Shanghai Sangon Biotech Co., Ltd. for chemical synthesis. Primer information is shown in Table 2.

[0051] Table 2 Primer sequence information

[0052]

[0053] Using expanded F-type mitochondria NAD2Gene-specific primers were used, and genomic DNA from gonadal tissues numbered 5, 6 (male), 7 (male), 9, and 10 (male) of known sex was used as a template. The PCR reaction system shown in Table 3 was followed by the following PCR reaction program using a PCR instrument: initial denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 45 seconds, annealing at 46℃ for 45 seconds, extension at 72℃ for 1 minute, for a total of 35 cycles; final extension at 72℃ for 10 minutes; and storage at 4℃.

[0054] Using expanded M-type mitochondria NAD2 Gene-specific primers were used, and genomic DNA from gonadal tissues numbered 5, 6 (male), 7 (male), 9, and 10 (male) of known sex was used as a template. The PCR reaction system shown in Table 3 was followed by the following PCR reaction program using a PCR instrument: initial denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 45 seconds, annealing at 57.1℃ for 45 seconds, extension at 72℃ for 1 minute, for a total of 20, 25, or 30 cycles; final extension at 72℃ for 10 minutes; storage at 4℃.

[0055] Table 3 PCR reaction system

[0056]

[0057] After amplification, the results of the amplification reaction were detected by agarose gel electrophoresis.

[0058] The specific procedure was as follows: a 0.8% agarose gel was prepared, and the electrophoresis apparatus parameters were set to 180V voltage, 160A current, and a duration of 20 minutes. After electrophoresis, the results were observed and recorded using a gel imaging system. The results obtained using an optical microscope were used as a control, and the M-type mitochondrial... NAD2 The amplification results and microscopic examination results of the genetically designed primer set were compared to verify the accuracy and reliability of sex identification.

[0059] Finally, the PCR amplification products were bidirectionally sequenced using Sanger sequencing to ensure sequence accuracy. The accuracy of sex determination was further confirmed by comparing the BLAST sequencing results from the National Center for Biotechnology Information (NCBI) with known sex-specific gene sequences.

[0060] 4. PCR reaction primer specificity test

[0061] By using M-type mitochondria NAD2Experiments using gene-specific primers for PCR amplification and agarose gel electrophoresis revealed that male samples successfully amplified bands in a PCR reaction program with 25 cycles, while female samples did not amplify any bands. Figure 1 Therefore, M-type mitochondria were chosen. NAD2 Genes were used to determine the sex of Manila clams, followed by amplification using a PCR reaction procedure with 25 cycles.

[0062] By using F-type mitochondria NAD2 Experiments using gene-specific primers for PCR amplification and agarose gel electrophoresis revealed that all 67 Manila clams amplified a band at the 600bp position. Figure 2 ).

[0063] In summary, the M-type mitochondria of the Manila clam NAD2 Gene sequences can be used as molecular markers to identify the sex of Manila clams. By using specific primers to amplify this molecular marker, the sex of Manila clams can be identified.

[0064] Example 2: Validation and Application of Molecular Markers

[0065] Using the DNA extracted from the Manila clam sample in Example 1 as an amplification template, nucleotide sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6 were used to amplify M-type mitochondria. NAD2 Gene-specific primers were used for PCR amplification to obtain PCR amplification products.

[0066] The results are as follows Figure 3 As shown, 26 samples amplified a band at the 600bp position, indicating these samples belonged to male individuals, while the remaining samples did not amplify this band at that position, indicating they belonged to female individuals. To ensure the accuracy of sex identification results, the PCR amplification results were compared with data obtained through microscopic observation. The results showed that the two methods were consistent. Finally, the PCR amplification products were sequenced using Sanger sequencing for bidirectional sequencing, and the sequencing results also matched the expected sex-specific sequence.

[0067] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a primer set of molecular markers for sex identification of Manila clams, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1; the primer set is a primer set specifically for detecting the molecular marker, and the primer set consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6; the application is for identifying the sex of Manila clams, breeding or assisted breeding of Manila clams.

2. The application according to claim 1, characterized in that, Gender identification is performed by detecting the presence or absence of the molecular marker described in claim 1.

3. A method for sex determination of Manila clams, characterized in that, Includes the following steps: DNA was extracted from the Manila clams to be tested; The DNA extracted from the Manila clam was amplified by PCR using the primer set described in claim 1 to obtain the PCR product. The PCR products were analyzed by electrophoresis. If the amplification product contains a 600bp fragment, the sex of the Manila clam being tested is male; if the amplification product does not contain a 600bp fragment, the sex of the Manila clam being tested is female.

4. The method for sex determination of Manila clams according to claim 3, characterized in that, Each 20 μL PCR amplification reaction system includes: 1 μL~2 μL DNA template, 10 μL 2×PCR Master mix, 0.1 μL~0.5 μL 10 μmol / L upstream primer, 0.1 μL~0.5 μL 10 μmol / L downstream primer, and deionized water to make up the difference.

5. The method for sex determination of Manila clams according to claim 3, characterized in that, The PCR amplification reaction procedure is as follows: (1) pre-denaturation; (2) denaturation; Annealing: 52.1℃~62.1℃, 45s; extension; 25 cycles in total; (3) terminal extension.

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