阅读说明：本技术 一种虾夷扇贝雌性特异性标记组合及应用 (Patinopecten yessoensis female specific marker combination and application ) 是由 张玲玲 郭振义 李亚娟 刘亮洁 王师 陆维 包振民 于 2019-10-16 设计创作，主要内容包括：本发明提供了一种虾夷扇贝雌性特异性分子标记组合及应用。本发明雌性特异性分子标记组合的核苷酸片段序列为SEQ ID NO:1和SEQ ID NO:4,上述序列可利用常规PCR扩增进行虾夷扇贝性别鉴定。本发明提供的两对雌性特异性引物,扩增产物大小分别为593bp和477bp。两对引物联合使用,对虾夷扇贝性别鉴定的准确率达100％。该性别检测方法简单,结果准确,对虾夷扇贝性别鉴定和性别调控的研究具有重要意义。(The invention provides a female specific molecular marker combination of Japanese scallops and application thereof. The nucleotide fragment sequences of the female specific molecular marker combination are SEQ ID NO. 1 and SEQ ID NO. 4, and the sequences can be used for carrying out sex identification on the Japanese scallops by utilizing the conventional PCR amplification. The two pairs of female specific primers provided by the invention have amplification product sizes of 593bp and 477bp respectively. The two pairs of primers are used together, and the accuracy rate of the sex identification of the patinopecten yessoensis reaches 100 percent. The sex detection method is simple, has accurate result, and has important significance for the research of sex identification and sex regulation of the patinopecten yessoensis.)

1. A female specific marker of Japanese scallop is characterized in that the nucleotide sequence of the female specific marker of Japanese scallop is SEQ ID NO. 1 and SEQ ID NO. 4; or the complementary sequences of SEQ ID NO 1, SEQ ID NO 4.

2. A product for sex determination of Japanese scallops, wherein the product is marked by the marker of claim 1.

3. The article of claim 2, wherein the article is a PCR amplification detection kit or a PCR amplification sequencing kit.

4. A method for detecting the sex of Japanese scallop, which is used for detecting the marker with the nucleotide sequence of SEQ ID NO. 1 and SEQ ID NO. 4 in claim 1.

5. The method according to claim 4, wherein the primer used for detecting the marker having the nucleotide sequence of SEQ ID NO. 1 has the sequence of SEQ ID NO. 2 as the upstream primer and SEQ ID NO. 3 as the downstream primer.

6. The method according to claim 4, wherein the primer used for detecting the marker having the nucleotide sequence of SEQ ID NO. 4 has the sequence of SEQ ID NO. 5 as the upstream primer and SEQ ID NO. 6 as the downstream primer.

Technical Field

The invention belongs to the technical field of sex identification of aquaculture animals, and particularly relates to a female specific marker combination of Japanese scallops and application thereof.

Background

Patinopecten yessoensis is an important economic shellfish in the class of gills, the family of scallops and the genus of scallops, and the annual value of the whole world exceeds $ 15 hundred million. The artificial breeding technology of the Japanese scallops is mature since 1982 in China, but the sex identification and sex control technology of the Japanese scallops is still deficient. The development of the researches on sex identification and sex regulation of the patinopecten yessoensis is beneficial to promoting the breeding process and has important significance for the development of the scallop breeding industry.

At present, the sex of the patinopecten yessoensis can be identified by three methods, namely a phenotype observation method, a tissue section method and a shellfish sex identification method based on sex differentiation gene expression. The limitation of the phenotype observation method is large, and the method is only suitable for identifying the sex of the bi-modal patinopecten yessoensis with obvious sex in the production season; the tissue observation method is complex to operate, and the sex identification success rate of the patinopecten yessoensis with gonads in resting periods is low; the method for identifying the sex of the patinopecten yessoensis based on the sex differentiation gene expression judges the sex by analyzing the expression quantity of the sex difference gene, and is suitable for the sex identification of the sex gland differentiated individuals.

The DNA molecular marker is an important tool for identifying sex, and is widely applied to species such as fish, shrimp, soft-shelled turtle and the like. Compared with gene expression and morphological methods, the method is not limited by tissues and development stages, the detection means is simple and quick, and the result is accurate. The method for identifying the sex of the patinopecten yessoensis is simple and effective to develop by developing the specific molecular marker of the sex of the patinopecten yessoensis, is low in cost, is beneficial to the development of the fine variety cultivation work of the patinopecten yessoensis, and has important significance for the research on the mechanism for determining the sex of the shellfish.

Disclosure of Invention

The invention aims to provide a female specific marker combination of Japanese scallops and application thereof, thereby making up the defects of the prior art.

The invention firstly provides a female specific marker combination of Japanese scallop, the nucleotide sequence of which is SEQ ID NO. 1 and SEQ ID NO. 4; or the complementary sequences of SEQ ID NO 1, SEQ ID NO 4.

The invention also provides a product for detecting the sex of the Japanese scallop, which is a mark for detecting that the nucleotide sequences are SEQ ID NO. 1 and SEQ ID NO. 4;

the product is preferably a PCR amplification detection kit or a PCR amplification sequencing kit;

the invention also provides a method for detecting the sex of Japanese scallop, which is used for detecting the markers with the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 4;

the primer is designed aiming at the sequence shown in SEQ ID NO. 1, wherein the sequence of the upstream primer of the primer is SEQ ID NO. 2, and the sequence of the downstream primer is SEQ ID NO. 3;

the primer designed aiming at the sequence shown in SEQ ID NO. 4, wherein the sequence of the upstream primer of the primer is SEQ ID NO. 5, and the sequence of the downstream primer is SEQ ID NO. 6.

The female specific marker combination provided by the invention can be used for carrying out sex identification on tissues except the gonads of the patinopecten yessoensis by utilizing the conventional PCR amplification, and the verification accuracy rate reaches 100%. Compared with the prior gene expression and morphological method, the technology has the advantages of simplicity, rapidness, accurate result and the like, and is beneficial to the development of sex-controlled breeding of the patinopecten yessoensis and the breeding industry.

Drawings

FIG. 1 is a diagram illustrating the result of the genetic sex identification of a female specific primer FSP1 of Patinopecten yessoensis in a Patinopecten yessoensis population. In the figure, the individuals with the numbers of 1-12 are female individuals and can amplify 593bp specific bands, the individuals with the numbers of 13-24 are male individuals and can not amplify bands, and M represents 2000bp DNA ladder.

FIG. 2 is a diagram showing the result of the genetic sex identification of the female specific primer FSP2 of Patinopecten yessoensis in the patinopecten yessoensis population. In the figure, the individuals with the numbers of 1-12 are female individuals and can amplify 477bp specific bands, the individuals with the numbers of 13-24 are male individuals and can not amplify bands, and M represents 100bp DNA ladder.

Detailed Description

The method for detecting the sex of the patinopecten yessoensis comprises 3 steps:

1) designing a female specific primer by taking the obtained female specific sequence as a template;

2) amplifying the genome DNA of the female and male patinopecten yessoensis by the synthesized female specific primers, checking the amplification efficiency and accuracy of the amplified genes, and optimizing an amplification system and the annealing temperature;

3) and (3) performing agarose electrophoresis on the amplification product obtained in the step (2), imaging and photographing an electrophoresis result by using a gel imaging instrument, and determining the sex of the patinopecten yessoensis through the amplification strip.

The invention is further illustrated by the following examples and figures.