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Macrobrachium rosenbergii sex specific molecular marker, screening method and application

A technology of Macrobrachium rosenbergii and molecular marker, applied in the field of genetic engineering, can solve the problem that crab sex markers have not obtained very reliable results and have not been further verified.

Inactive Publication Date: 2012-10-10
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies on sex markers in crabs have not yet achieved very reliable results
Qiu Tao used RAPD to analyze male and female populations and individuals of Chinese mitten crabs. Although male-specific candidate markers were obtained, they were not further verified (Qiu Tao, Lu Renhou, Xiang Chaomei, Zhang Jing, Xie Hao. Using RAPD Technical identification of male and female differences in Chinese mitten crabs. Acta Aquatica Sinica, 1998, 2, 175-177)
Wang Yilei used AFLP technology to screen out the male and female difference fragments of Scylla serrata, which proved that there are differences in DNA sequences between the male and female genomes, but did not form these difference fragments into real molecular markers (Wang Yilei, Dai Jun, Yao Yanglie, Zhang Ziping. Using AFLP technology to screen male and female differential DNA fragments of Scylla serrata. Chinese Fisheries Science. 2004, 4, 286-290)

Method used

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  • Macrobrachium rosenbergii sex specific molecular marker, screening method and application
  • Macrobrachium rosenbergii sex specific molecular marker, screening method and application
  • Macrobrachium rosenbergii sex specific molecular marker, screening method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1. Using the AFLP method to obtain sex-specific molecular markers in Macrobrachium rosenbergii

[0102] Materials: Macrobrachium rosenbergii, 39 females and 39 males, were purchased from the orchard farmer’s market in Pudong New District, Shanghai. The muscles from the tail fan of Macrobrachium rosenbergii were taken, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

[0103] Differentiation of male and female Macrobrachium rosenbergii:

[0104] Macrobrachium rosenbergii is dioecious, and the characteristics of both sexes can be distinguished in appearance with the naked eye:

[0105] The female shrimp is smaller than the male shrimp; the mature female shrimp has well-developed ovaries, and the orange-yellow ovaries of the female shrimp can be seen through the carapace.

[0106] The second pair of legs of the male is particularly large, much longer than the length of the body, and is blue in color; the inner limb of the second swimming leg o...

Embodiment 2

[0162] Example 2, SCAR verification of the genome-specific sequence of Macrobrachium rosenbergii

[0163] 3 Verification of sex-specific molecular markers in Macrobrachium rosenbergii

[0164] 3.1 SCAR verification of sex-specific molecular markers in Macrobrachium rosenbergii

[0165] According to the sequencing results of the sex-specific molecular marker (MaroF300) of Macrobrachium rosenbergii, a pair of specific SCAR primers Scar202F and Scar202R were designed after optimization by Primer Premier5.0 design software: their sequences are respectively SEQ ID NO: 24: (F: 5' -GGGCAATGATGACTGACT-3'), SEQ ID NO: 25 (R: 5'-TTAGCGCCTGATGGTATG-3').

[0166] Macrobrachium rosenbergii genomic DNA was amplified by PCR with SCAR primers.

[0167] The primers for PCR were SCAR primers Scar202F and Scar202R (concentration: 10uM).

[0168] The reaction system is 10×PCR Buffer 2.5μl; 25mM MgCl 2 1.5μl; 2.5mM dNTP 2μl, Scar202F and Scar202R primers 1μl each, Taq DNA polymerase 1.5U, mal...

Embodiment 3

[0172] Example 3 Application of Macrobrachium rosenbergii sex-specific molecular markers

[0173]The sex-specific molecular marker of Macrobrachium rosenbergii can be used to identify the sex of Macrobrachium rosenbergii, comprising the following steps:

[0174] (1) Extract the genomic DNA of Macrobrachium rosenbergii to be tested;

[0175] (2) Prepare specific SCAR primers Scar202F and Scar202R: their sequences are SEQ ID NO: 24:, SEQ ID NO: 25; synthesized from Shanghai Sangon Bioengineering Co., Ltd.

[0176] (3) PCR amplification of Macrobrachium rosenbergii genomic DNA with the SCAR primers;

[0177] The primers for PCR were SCAR primers Scar202F and Scar202R (concentration: 10uM).

[0178] The reaction system is 10×PCR Buffer 2.5μl; 25mM MgCl 2 1.5μl; 2.5mM dNTP 2μl, Scar202F and Scar202R primers 1μl each, Taq DNA polymerase 1.5U, male and female individual DNA template 1ul, make up to 25μl with sterile water.

[0179] The reaction program was pre-denaturation at 94...

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Abstract

The invention discloses a macrobrachium rosenbergii sex specific molecular marker, in a genome of macrobrachium rosenbergii, a segment of a DNA fragment of 300bp is separated, its sequence is SEQ ID NO: 23, and the segment can be used for detecting the sex of macrobrachium rosenbergii. The screening method for the sex specific molecular marker comprises the following steps: extracting the macrobrachium rosenbergii DNA, performing an AFLP analysis, cloning the sex specific fragment, designing a specific primer, detecting the feasibility of the sex specific fragment and the like. The screened macrobrachium rosenbergii sex specific molecular marker establishes a method for identifying the genetic sex of macrobrachium rosenbergii, the obtained molecule marker enables accurate and stable detection of macrobrachium rosenbergii at the early growth stage and undetermined sex on the appearance, and is in favor of establishment of a monosex cultivation system for increasing the yield. A female specific fragment obtained in the genome of macrobrachium rosenbergii provides a necessary condition for positioning on chromosome.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. The invention relates to the specific sequence of the female genome of Macrobrachium rosenbergii, specifically, a sex-specific molecular marker of Macrobrachium rosenbergii, a screening method and application thereof. Background technique [0002] Macrobrachium rosenbergii (Macrobrachium rosenbergii), known as the king of freshwater shrimp, is concentrated in the coast of Ecuador. It is one of the three largest shrimp species in the world. Its shell is thin and fat, and its meat is tender and delicious. full of nutrition. Since it was introduced into China from Japan in 1976, it has been promoted in many provinces (cities, districts) of 10 in the south. The economic benefits are considerable, and it occupies an important position in my country's freshwater aquaculture. [0003] Macrobrachium rosenbergii is dioecious, and the growth rate of males is 10-20% higher than that of females,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 丘高峰江学慧吕慧锋
Owner SHANGHAI OCEAN UNIV