Sturgeon sexuality difference molecular marker and application thereof

A molecular marker, differential technology, applied in DNA/RNA fragmentation, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of individual size limitation, death of sturgeon, and high requirements for operating experience, and achieves convenient operation and method. Accurate and reliable, the effect of improving economic efficiency

Active Publication Date: 2016-08-17
深圳华大海洋科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Minimally invasive surgery is a routine method for determining the sex of sturgeon. First, anesthetize the sturgeon, then use a scalpel to make a small incision between the 4th and 5th ventral plates in front of the pelvic fin, and take out the gonad tissue with the naked eye under a microscope. Observe and identify the sex or gonad development period. This method is likely to cause the death of the sturgeon, and the individual sturgeon must have gonad development, and requires a high level of operator experience.
The endoscopic technique is to insert the speculum shaft from the genital opening of the sturgeon so that the front part is close to the gonad tissue, and observe through the eyepiece or an external imaging system. This method is also limited by the size of the individual, and the identification rate is low, requiring high operating experience.
Ultrasonic identification method is to use ultrasonic means to capture images of internal organs of sturgeon, and then analyze the results, affected by the obstruction of abdominal tissue, the identification rate is also low, and the different degrees of gonad development also affect the results
The blood biochemical and hormone index method is based on the fluctuation of certain hormones and biochemical indicators during the gonad development period of the sturgeon to detect the sex of the sturgeon. This method is unstable, there are many indicators to be discussed, and the detection procedure is complicated

Method used

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  • Sturgeon sexuality difference molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Obtaining Molecular Markers Related to Sturgeon Sex

[0027] 1.1 Acquisition of sexed sturgeon populations

[0028] The population used was Sturgeon's sturgeon with known sex from a sturgeon farm in Jiangsu, with 50 male and 50 fish each. The cut dorsal fin rays of the fish were stored in 95% ethanol at -20°C for genomic DNA extraction.

[0029] 1.2 Genomic DNA extraction from sturgeon

[0030] In this experiment, the conventional phenol-chloroform method was used to extract genomic DNA from sturgeon fin rays, and the specific steps were as follows:

[0031] (1) Take 0.3-0.5g of fin rays in a 1.5ml Eppendorf tube, cut it into pieces, and dry it for 20 minutes on a clean bench;

[0032] (2) After the ethanol is basically volatilized, wash with TE buffer solution (10mmol / ml Tris, 1mmol / ml EDTA, SDS 5%, pH=8.0) for 1-2 times, then add 600μl DNA extraction solution (0.001mol / LTris- C1, 0.1mol / L EDTA, SDS 5%, pH=8.0) and 3μl proteinase k (200mg / ml), digest in a...

Embodiment 2

[0037] Example 2 Verification of Molecular Markers Related to Sturgeon Sex

[0038] 2.1 Extraction of genomic DNA from fin rays of sturgeon to be verified

[0039] Sturgeons to be verified belong to the group in Example 1, 12 males and females were randomly selected, and the DNA extraction method was as described in Example 1.

[0040] 2.2 Amplification of nucleotide fragments containing male-specific reads

[0041] Primers were designed respectively according to the DNA sequences SEQ ID NO.4 and SEQ ID NO.5 of the male-specific reads, and the primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3. Using the genomic DNA in 2.1 as a template, the PCR reaction system in 25 μl is: 50-100ng / μl template DNA 1μl, 10pmol / μl primers F and R 1μl each, 10mmol / L dNTP mix 2.0μl, 5U / μl Taq DNA polymerase 0.125 μl, 2.5 μl of 10×PCR reaction buffer, and double-distilled water as the balance; PCR reaction conditions: 94°C for 5 minutes; 30 cycles of 94°C for 30 seconds, 55°C for 30 secon...

Embodiment 3

[0042] Example 3 Application of Molecular Markers Related to Sturgeon Sex

[0043] 3.1 Extraction of genomic DNA from the fin rays of the sturgeon to be tested

[0044] The sturgeons to be tested were Sturgeon's sturgeons of known sex from a sturgeon farm in Zhejiang, 12 males and 12 males were randomly selected, and the DNA extraction method was as described in Example 1.

[0045] 3.2 Amplification of sex-related molecular markers in sturgeon

[0046] Use the primers whose sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, use the genomic DNA in 3.1 as a template, and use the PCR reaction system and conditions in 2.2. The results showed that the amplified band of 286bp was male fish, while the female fish had no such amplified product. The agarose gel electrophoresis pattern of the amplified product is attached figure 2 As shown, the middle lane of the gel map is the molecular weight Marker, the uppermost band of the Marker is 1500bp, the lowermost band is 100bp, and the...

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Abstract

The invention discloses a sturgeon sexuality difference molecular marker and an application thereof; the marker has the nucleotide sequence shown in SEQ ID NO.1; a DNA sequence shown in the SEQ ID NO.1 is detected by PCR primers, and according to a PCR detection result, the sturgeon sexuality is identified; a forward primer F of the PCR primers is 5'-CACACTGATGCTCTACATGT-3', a reverse primer R is 5'-AGGCTGGACAGTGATGCTGT-3', and the forward primer F and the reverse primer R are respectively shown in SEQ ID NO.2 and SEQ ID NO.3. The invention provides a nucleic acid fragment for detecting the sturgeon sexuality and the PCR primers for detecting the molecular marker related to sturgeon; the molecular marker is not limited by the age of the sturgeon, is accurate and reliable, is easy to operate, and has important application value in production.

Description

technical field [0001] The invention relates to a sturgeon sex difference molecular marker and its application. Background technique [0002] Sturgeon is a large cartilaginous scale fish with high nutritional and economic value. It can also be used as an ornamental fish. It takes a long time to sexually mature, generally at least 5 years, and some even as long as 15 years. Sturgeon has no secondary sex. There is only one "same" gonad in the juvenile stage, and distinguishing the sex of sturgeon is of great significance to the breeding and processing industry of sturgeon. [0003] At present, the sex identification methods of sturgeon mainly include minimally invasive surgery, endoscopic technology, ultrasonic identification, blood biochemical and hormone index methods. Minimally invasive surgery is a routine method for determining the sex of sturgeon. First, anesthetize the sturgeon, then use a scalpel to make a small incision between the 4th and 5th ventral plates in front...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 石琼游欣欣徐军民阮志强马兴宇
Owner 深圳华大海洋科技有限公司
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