Specific marker for male and female genetic sex of oplegnathus punctatus, identification method and kit

A porphyra, specific technology, applied in the field of specific markers and identification methods and kits for the genetic sex of porphyra male and female, can solve the problems of unstable amplification products, inconsistent amplification efficiency of target bands, confusion, etc. , to achieve rapid and accurate identification and improve the genetic breeding process of porpoise

Active Publication Date: 2022-03-01
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

[0004] The reported genetic sex identification of the male and female porcupine adopts the "three-body method", that is, the combined amplification analysis of three primers. Because the annealing temperature of the three primers used in this method is different, the amplification efficiency of the target band is inconsistent. , The amplified product is unstable; at the same time, the size of the genetic male-specific target band designed by this method is 222 bases (bp), which is easily confused with the primer-dimer (10~250bp) generated during the PCR amplification process ; In addition, because the difference between the other two target bands of male individuals is only 12 bases (bp) in this method, the discrimination between the two target bands on conventional agarose gel electrophoresis is poor

Method used

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  • Specific marker for male and female genetic sex of oplegnathus punctatus, identification method and kit
  • Specific marker for male and female genetic sex of oplegnathus punctatus, identification method and kit
  • Specific marker for male and female genetic sex of oplegnathus punctatus, identification method and kit

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Screening and verification of male and female shared and male-specific DNA markers

[0034] Discovery of hermaphrodite Y chromosome with a large insertion target DNA sequence:

[0035] The DNA sequence of the heteromorphic chromosome (neoY) of the male fish used in the present invention and the X of the female fish 1 Chromosomal DNA sequence, from the Institute of Oceanology, Chinese Academy of Sciences, Marine Fish Aquaculture and Breeding Technology Laboratory Li Jun's research team entrusted Guangzhou Kidio Biotechnology Co., Ltd. to complete the female and male porpoise using the third-generation PacBio whole-genome sequencing technology. The genome was sequenced and assembled, and the assembly results were basically consistent with the published genome information of the grouper (CNP0001488, Li et al., 2021). Using comparative genomic bioinformatics to analyze the genome sequences of the male and female porpoise visible (see figure 1 ), Porpoise female...

Embodiment 2

[0043] Example 2 Establishment and Application of Genetic Sex Determination Technology for Male and Female Porpoise

[0044] Genetic sex identification was carried out on the grouper bream from Laizhou Mingbo Aquatic Products Co., Ltd. in Laizhou, Yantai, Shandong (among them, 12 phenotypes were female and 12 phenotypes were male):

[0045] High-quality DNA extraction: use the Tiangen Marine Animal DNA Extraction Kit to extract the DNA from the fin ray of the grouper, use 0.8% agarose gel electrophoresis to identify the integrity of the genomic DNA, and use a UV spectrophotometer to measure the OD value of the DNA supernatant. Adjust the DNA concentration to 60ng / µL and freeze it at -20°C for later use.

[0046] PCR reaction system and Touch down PCR amplification identification: The sex-specific DNA fragment primers ChXY_F: 1 and ChXY_R: 2 were used to detect the genetic sex of the red sea bream by PCR. PCR reaction system 25µL: 10×Buffer 5.0µL, dNTP 4.0µL, rTaq enzyme (5U / µ...

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Abstract

The invention relates to a qualitative detection method for species germplasm identification by a molecular biological technology, in particular to a specific marker for male and female genetic sex of oplegnathus punctatus, an identification method and a kit. The nucleotide sequences of the specific markers for the chromosomes of the female fish and the male fish of the oplegnathus punctatus are bases shown as SEQ ID NO: 1 (Xfm ID Nm: 1) and SEQ ID NO: 2 (Ym ID Nm: 1). According to the method, a pair of primers is utilized to amplify two DNA fragments of 635bp and 925bp with the difference of 290bp in a genetic male individual, only a single DNA fragment of 635bp is amplified in a genetic female individual, and agarose gel electrophoresis distinguishing can be carried out, so that the time for accurately identifying the genetic sex of the oplegnathus punctatus is shortened, and the sex detection efficiency is improved. The method has important significance and application value in sex identification, high-male offspring seed preparation and family breeding of oplegnathus punctatus.

Description

technical field [0001] The invention relates to a qualitative detection method for species germplasm identification by molecular biology technology, in particular to a specific marker and identification method and a kit for the genetic sex of porpoise male and female. Background technique [0002] Porpoise belongs to the order Perciformes, family Oplegnathidae, genus Perciformes ( Oplegnathus ), mainly distributed in the warm and tropical waters of the western Pacific Ocean, prefers to inhabit rocky reefs, gravel, and algae-rich sea areas at a water depth of 10-100m, and is a reef-loving bottom-dwelling fish; juvenile fish have the ability to drift with seaweed in summer and autumn ecological habits. Carnivorous, with sharp teeth, can crush hard shells such as shellfish or sea urchins (Meng Qingwen et al., 1995). The sapphire skin is rich in collagen, a variety of amino acids and unsaturated fatty acids. It has a unique taste and is known as "the best sashimi" in Japanese ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6879C12N15/11
CPCC12Q1/6888C12Q1/6879
Inventor 肖永双马玉婷李军肖志忠吴燕铎赵海霞马道远
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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