CN112176049A - DNA molecular marker for identifying sex of apostichopus japonicus living body and identification method - Google Patents

Abstract

The invention discloses a DNA molecular marker for identifying the sex of an apostichopus japonicus living body and an identification method, wherein the nucleotide sequence of the DNA molecular marker is shown as SEQ ID NO: 1, the identification method comprises the following steps: collecting apostichopus japonicus canal feet, and extracting apostichopus japonicus DNA; and (3) carrying out PCR reaction by using the obtained DNA as a template, wherein the nucleotide sequences of the upstream primer and the downstream primer of the PCR reaction are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the PCR reaction system is that a template accounts for 30-100 ng, 2X PCR mix accounts for 10 muL, an upstream primer accounts for 1 muL, a downstream primer accounts for 1 muL, and sterilizing water is supplemented to 20 muL; the PCR reaction program is denaturation at 98 ℃ for 30 s; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 15s, extension at 72 deg.C for 1min, and performing 30 cycles; extending for 5min at 72 ℃; storing the PCR reaction product at 4 ℃; and (3) carrying out electrophoresis detection on the PCR reaction product, and amplifying to obtain a specific band with the length of 314bp, wherein the specific band is a male individual, and the specific band is a female individual otherwise.

Description

DNA molecular marker for identifying sex of apostichopus japonicus living body and identification method

Technical Field

The invention belongs to the technical field of aquaculture sex identification, and particularly relates to a DNA molecular marker for identifying the sex of an apostichopus japonicus living body and an identification method.

Background

The apostichopus japonicus has extremely high economic value, is the first of eight delicacies of marine products, is one of mariculture varieties, and is an indispensable link for distinguishing the sex of the apostichopus japonicus in the processes of cultivating the apostichopus japonicus and the like. However, apostichopus japonicus is a female-male variant and generally develops to sexual maturity in two years, but the gonads of the female and male are not identified by naked eyes in an undifferentiated stage, a resting stage, a proliferation stage (recovery stage), a growth stage, a maturation stage and a discharge stage, and must be identified by a specific method. At present, most of sex identification methods for apostichopus japonicus require dissection to obtain gonads, and cannot meet the requirements of in vivo identification through histological sections or quantitative analysis of gene expression. Although there are biopsy methods and PAGE electrophoresis methods using body fluid protein extracts, the procedures are complicated and the accuracy is low. The DNA molecular marker is an important tool for identifying the sex of the living body, is not limited by tissues and development stages, can realize identification by using a PCR operation technology, and has the advantages of simple and quick operation, accurate result and the like. However, no relevant report for identifying the sex of the apostichopus japonicus living body based on DNA molecular markers exists so far.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a DNA molecular marker for identifying the sex of an apostichopus japonicus living body and an identification method.

The technical solution of the invention is as follows: a DNA molecular marker for identifying the sex of an apostichopus japonicus living body is characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

The identification method based on the DNA molecular marker for identifying the sex of the apostichopus japonicus living body is characterized by comprising the following steps of:

a. collecting apostichopus japonicus canal feet, and extracting apostichopus japonicus DNA;

b. using the obtained DNA as a template to carry out PCR reaction, wherein upstream and downstream primers of the PCR reaction

The nucleotide sequences of (a) are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the PCR reaction system is that a template accounts for 30-100 ng, 2X PCR mix accounts for 10 muL, an upstream primer accounts for 2 mumol and 1 muL, a downstream primer accounts for 2 mumol and 1 muL, and sterilized water is supplemented to 20 muL; the PCR reaction program is denaturation at 98 ℃ for 30 s; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 15s, extension at 72 deg.C for 1min, and performing 30 cycles; extending for 5min at 72 ℃; storing the PCR reaction product at 4 ℃;

c. and (3) carrying out electrophoresis detection on the PCR reaction product, and amplifying to obtain a specific band with the length of 314bp, wherein the specific band is a male individual, and the specific band is a female individual otherwise.

According to the invention, the sex identification of the living body can be realized only by collecting a small amount of tissue of the angiopodium of the apostichopus japonicus, the accuracy rate reaches 100%, the growth state and the survival rate have no obvious difference compared with a control group, and the method has the advantages of simple and rapid detection, high accuracy rate and the like.

Drawings

FIG. 1 is a diagram showing the results of electrophoretic detection of PCR reaction products of a sample according to an embodiment of the present invention.

Fig. 2 is a diagram showing the result of the histological determination of the gonad of the sample according to the embodiment of the present invention.

Detailed Description

The method adopts a classical phenol chloroform method to extract genome DNA of twelve female apostichopus japonicus canal feet and genome DNA of twelve male apostichopus japonicus canal feet, uses an Illumina Hiseq Xten sequencing platform and a 2bRAD sequencing technology to carry out sequencing, uses Pear software to carry out sequence splicing, and respectively obtains the genomes of the female apostichopus japonicus and the male apostichopus japonicus. By comparing the female and male apostichopus japonicus genomes, a sequence is found to exist only in a male individual and not in a female individual, so that the nucleotide sequence shown as SEQ ID NO: 1, the DNA molecular marker for identifying the sex of the apostichopus japonicus living body.

The identification method based on the DNA molecular marker for identifying the sex of the apostichopus japonicus living body is characterized by comprising the following steps:

a. randomly collecting 24 small apostichopus japonicus tube feet (DNA extraction is only required), and extracting the apostichopus japonicus tube foot DNA by a phenol chloroform extraction method;

b. using the obtained DNA as a template to carry out PCR reaction, wherein upstream and downstream primers of the PCR reaction

The nucleotide sequences of (a) are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the PCR reaction system is that a template is 30-100 ng, 2X PCR mix is 10 muL, an upstream primer (2 mumol) is 1 muL, a downstream primer (2 mumol) is 1 muL, and sterilized water is supplemented to 20 muL; the PCR reaction program is denaturation at 98 ℃ for 30 s; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 15s, extension at 72 deg.C for 1min, and performing 30 cycles; extending for 5min at 72 ℃; storing the PCR reaction product at 4 ℃;

c. preparing 1% agarose gel for electrophoresis detection to perform electrophoresis detection on PCR reaction products, wherein the electrophoresis results are shown in figure 1 as two types: the left side is male with a specific band of 314bp in length, the right side is female without a specific band of 314bp in length, and M represents a 2 kb DNA ladder. The identification result is 12 each of the male individuals and the female individuals in 24 apostichopus japonicus.

Experiment:

1. the tested individuals identified by the invention and a control group (24 apostichopus japonicus selenka without collecting tube feet) are bred for one month under the same condition, and the growth state and the survival rate of the tested individuals are not obviously different from those of the control group.

2. Respectively placing the gonads of the tested individuals fed in one month into 4% paraformaldehyde for fixation, and using the gonads for histological judgment; fixing with 4% paraformaldehyde overnight, pouring out the fixing solution, washing with PBS for three times, then permeating with 30% sucrose at room temperature for 2-3 hours, embedding gonadal tissue blocks with an OCT embedding machine, carrying out frozen section with a freezing microtome, staining with eosin/hematoxylin, and taking a picture under a microscope to judge the sex of the gonadal of the obtained sea cucumber. As shown in FIG. 2, the results of the 24 pictures were female numbers 1 to 12 and male numbers 1 to 12, respectively. The result shows that the accuracy rate of the apostichopus japonicus living body sex identification method provided by the embodiment of the invention is 100%.

Sequence listing

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Claims (2)

1. A DNA molecular marker for identifying the sex of an apostichopus japonicus living body is characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. A method for identifying DNA molecular markers for identifying the sex of Apostichopus japonicus living bodies according to claim 1, which comprises the following steps:

a. collecting apostichopus japonicus canal feet, and extracting apostichopus japonicus DNA;

b. using the obtained DNA as a template to carry out PCR reaction, wherein upstream and downstream primers of the PCR reaction

The nucleotide sequences of (a) are respectively shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the PCR reaction system is that a template accounts for 30-100 ng, 2X PCR mix accounts for 10 muL, an upstream primer accounts for 2 mumol and 1 muL, a downstream primer accounts for 2 mumol and 1 muL, and sterilized water is supplemented to 20 muL; the PCR reaction program is denaturation at 98 ℃ for 30 s; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 15s, extension at 72 deg.C for 1min, and performing 30 cycles; extending for 5min at 72 ℃; storing the PCR reaction product at 4 ℃;

c. and (3) carrying out electrophoresis detection on the PCR reaction product, and amplifying to obtain a specific band with the length of 314bp, wherein the specific band is a male individual, and the specific band is a female individual otherwise.

Images