DNA molecular marker for identifying sex of living strongylocentrotus intermedius and identification method

Abstract

The invention discloses a DNA molecular marker and an identification method for identifying the sex of a living body of strongylocentrotus intermedius, the nucleotide sequence of the DNA molecular marker is as shown in SEQ ID NO: 1, and the identification method is carried out according to the following steps: cutting the foot of the strongylocentrotus intermedius, and extracting the genome DNA of the strongylocentrotus intermedius; the obtained genome DNA is used as a reaction template, a PCR amplification reaction is carried out, and nucleotide sequences of upstream and downstream primers of the PCR amplification reaction are shown as SEQ ID NO: 2 and SEQ ID NO: 3 respectively; and carrying out agarose gel electrophoresis detection on a PCR reaction product, determining that a specific band with the length of 120bp is amplified as a female individual, and determining that a specific band without an obvious band is a male individual.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture sex identification technology, and particularly relates to a DNA molecular marker and identification method for sex identification of live sea urchins. Background Technology

[0002] Middle ball sea urchin ( Strongylocentrotus intermedius As an economically valuable species, sea urchins are widely found in the coastal areas of Liaoning, Shandong, and Guangdong provinces of my country. The gonads, the only edible part of the sea urchin, are rich in unsaturated fatty acids, a large amount of essential amino acids, and small-molecule active substances, possessing extremely high nutritional and medicinal value. Studies have shown that sea urchins of different sexes exhibit significant differences in gonad quality, individual development, growth rate, and immune capacity. In recent years, with the continuous increase in market demand and the rise of the sea urchin aquaculture industry, wild sea urchin resources have declined. Cultivating superior, stress-resistant new strains and realizing sex-controlled breeding and monosex aquaculture models are current challenges and future research directions for the sea urchin aquaculture industry. Currently, most sea urchin sex identification requires dissection to obtain gonadal tissue, followed by histological morphological observation or quantitative gene expression analysis to determine genetic sex, which cannot meet the needs of live identification. In the case of *Spinycticebus glaber* (a type of sea urchin),... Mesocentrotus nudus In this study, DNA sex-specific molecules enable rapid and accurate identification of the genetic sex of live *Echinochloa glabripennis*, unaffected by developmental stage or environmental factors. Therefore, searching for sex-specific molecular markers in *Echinochloa glabripennis* provides support for accurate identification of the genetic sex of live *Echinochloa glabripennis*, as well as for new models of sex-controlled breeding and asexual breeding. Summary of the Invention

[0003] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a DNA molecular marker and identification method for sex determination of live sea urchins.

[0004] The technical solution of the present invention is: a DNA molecular marker for sex identification of live intermediate sea urchins, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0005] A method for sex determination of live sea urchins based on the aforementioned intermediate spherical sea urchin DNA molecular markers, comprising the following steps: a. Collect the tube feet of live sea urchins and extract their genomic DNA; b. Using the genomic DNA of the sea urchin *Ulva intermedius* as a template, PCR amplification was performed. The nucleotide sequences of the upstream and downstream primers for the PCR amplification reaction are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The total PCR amplification reaction volume was 20 µL, and the specific reaction volume was as follows: 2 µL template, 10 µL 2×Es Taq Master Mix, 0.8 µL upstream primer, 0.8 µL downstream primer, and 6.4 µL enzyme-free water. The PCR amplification reaction program was as follows: denaturation at 95 °C for 2 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; extension at 72 °C for 2 min; the product was stored at 4 °C after amplification. c. The PCR amplification products were detected by agarose gel electrophoresis. Individuals with a specific band of 120 bp were female, while those without a clear band were male.

[0006] This invention allows for live sex identification of *Ulva intermedius* by simply harvesting a small amount of tube foot tissue, achieving a 100% accuracy rate. After harvesting the tube feet, the experimental group showed no significant difference in growth status and survival rate compared to the control group. This method is simple, rapid, and highly reliable. Attached Figure Description

[0007] Figure 1 This is a graph showing the results of agarose gel electrophoresis detection of the PCR amplification reaction products in an embodiment of the present invention.

[0008] Figure 2 This is a diagram showing the results of histological determination of the gonadal tissue in an embodiment of the present invention. Detailed Implementation

[0009] This invention employs the classic phenol-chloroform method to extract genomic DNA from the tube feet of 15 male and 15 female *Ulva intermedius*. Genome resequencing is then performed using the Illumina Hiseq Xten sequencing platform. Based on the sequencing results, sex-specific molecular marker analysis is conducted to screen for sex-differentiated DNA molecular tags. Comparison revealed that a 120bp sequence (as shown in SEQ ID NO: 4) exists only in female individuals and not in males. Therefore, a 120bp sequence (as shown in SEQ ID NO: 1) as a live sex marker for *Ulva intermedius* was invented.

[0010] Based on the DNA molecular marker-based identification method for sex determination in live sea urchins described above, the following steps are performed sequentially: a. Cut the tube feet of 20 live sea urchins and extract genomic DNA from them using a kit method; b. Using the genomic DNA of the intermediate sea urchin obtained in step a as a template, PCR amplification was performed. The nucleotide sequences of the upstream and downstream primers in the PCR reaction are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, i.e., SEQ ID NO: 2 is the upstream primer and SEQ ID NO: 3 is the downstream primer. The total PCR amplification reaction volume was 20 µL, and the specific reaction volume was as follows: 2 µL genomic DNA template, 10 µL 2×Es Taq Master Mix, 0.8 µL upstream primer, 0.8 µL downstream primer, and 6.4 µL enzyme-free water. The PCR amplification reaction program was as follows: 95 °C pre-denaturation for 2 min; 95 °C denaturation for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 30 s, for 35 cycles; 72 °C extension for 2 min; after amplification, the product was stored at 4 °C. c. Prepare a 1% agarose gel for electrophoresis detection of the PCR amplification products in b. The electrophoresis results are as follows: Figure 1 As shown: the specific band amplified by the female marker is 120 bp in length, and M represents a 2000 bp DNA Marker; individuals amplified with a specific band of 120 bp in length are female, while those without a clear band are male.

[0011] The results showed that 10 of the 20 intermediate sea urchins were female and 10 were male, numbered as female 1-10 and male 1-10 respectively.

[0012] Histological identification of the gonads of the intermediate globule sea urchin: a. Twenty individuals identified by this invention and a control group (20 intermediate sea urchins without tube feet collection) were raised under the same conditions for one month. Their growth status and survival rate were not significantly different from those of the control group.

[0013] b. Gonadal tissues from the above 20 individuals were incubated overnight at 4°C in 4% paraformaldehyde (PFA) fixative for the following gonadal histological examination. The specific method is as follows: On the second day, the tissues were washed three times with phosphate buffered saline (PBS) for 15 min each time to remove any remaining PFA solution. Then, 30% sucrose solution was added, and the tissues were washed twice for 15 min each time. The tissues were then infiltrated overnight at 4°C in 30% sucrose solution. On the third day, the tissues were embedded in OCT cryoemulation medium. Gonadal tissue sections with a thickness of 5 μm were obtained using a Leica CM 1900-1-1 cryostat. The sections were stained with eosin and hematoxylin-eosin staining (HE), photographed under a microscope, and the genetic sex of the sea urchins was determined. The results are as follows: Figure 2 As shown, the 20 images are females numbered 1 to 10 and males numbered 1 to 10, indicating that the accuracy of the live sex identification method for the intermediate sea urchin in this embodiment of the invention is 100%.

Claims

1. A DNA molecular marker for sex identification in live sea urchins, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

1.

2. A method for sex determination of live sea urchins based on the DNA molecular markers described in claim 1, characterized in that... Follow these steps in sequence: a. Collect the tube feet of live sea urchins and extract their genomic DNA; b. Using the genomic DNA of the sea urchin *Ulva intermedius* as a template, PCR amplification was performed. The nucleotide sequences of the upstream and downstream primers for the PCR amplification reaction are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The total PCR amplification reaction volume was 20 µL, and the specific reaction volume was as follows: 2 µL template, 10 µL 2×Es Taq Master Mix, 0.8 µL upstream primer, 0.8 µL downstream primer, and 6.4 µL enzyme-free water. The PCR amplification reaction program was as follows: denaturation at 95 °C for 2 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; extension at 72 °C for 2 min; the product was stored at 4 °C after amplification. c. The PCR amplification products were detected by agarose gel electrophoresis. Individuals with a specific band of 120 bp were female, while those without a clear band were male.

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