Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof

A Chinese mitten crab and molecular marker technology, which is applied in the fields of molecular biology and genetic breeding, can solve the problems that have not yet been seen in the growth traits of Chinese mitten crab, and achieve the effect of clear selection goals

Inactive Publication Date: 2014-03-26
天津市水生动物疫病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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Method used

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  • Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof
  • Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof
  • Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0018] Example 1

[0019] Measurement and Record of Phenotypic Values ​​of Population Growth Traits of Qilihai River Crab in F6 Generation

[0020] Randomly select 60 male and female individuals from the sexually mature group of river crabs, and record them one by one;

[0021] Measure the main growth traits of body length, body width, body height, IV step length (unit: mm) and weight (unit: g) indicators, record the individual's gender, the data is shown in Table 1.

[0022] Table 1 Phenotypic values ​​of population growth traits of Eriocheir sinensis

[0023] Numbering

Example Embodiment

[0024] Example 2. Extraction of muscle total DNA of F6 generation breeding population of Eriocheir sinensis (cut one foot for each river crab)

[0025] Using phenol-chloroform method to extract total muscle DNA, the specific steps are as follows:

[0026] 1) Cut 100mg of frozen muscle sample, chop it into 1.5ml sterile centrifuge tube;

[0027] 2) Add 500μl extraction buffer (6M urea, 10mM Tris-HCl, 125mM NaCl, 1% SDS, 10mM EDTA, pH7.5), 8μl proteinase K (20mg / ml), digest overnight at 37℃;

[0028] 3) After the sample is completely digested, add an equal volume of PCI (phenol:chloroform:isoamyl alcohol=25:24:1), spin and stir for 20min, centrifuge at 5000rpm for 15min at room temperature, and take the supernatant in another new sterile centrifuge tube in. Repeat PCI extraction 3 times;

[0029] 4) Carefully transfer the supernatant to a new centrifuge tube, add an equal volume of CI (chloroform: isoamyl alcohol = 24:1), rotate and stir for 10 minutes, centrifuge at 5000 rpm for 15 min...

Example Embodiment

[0035] Example 3. Development of EST-SSR molecular markers

[0036] 1) Log in to the NCBI database (http: / / www.ncbi.nlm.nih.gov / dbEST) to search for the EST sequence of Chinese mitten crab, online (http: / / www.gramene.org / db / searches / ssrtool) to find CDNA fragments of microsatellite sequence, the standard is to repeat more than 5 times (including 5 times);

[0037] 2) According to the sequencing results and EST database screening results, select suitable DNA fragments containing microsatellite sequences, use Primer Premier 5.0 (http: / / www.premierbiosoft.com / ) software to design primers, and send them to Shanghai Shenggong for synthesis. The theoretical parameters considered for primer design include: GC content, annealing temperature, mismatches, primer dimer, hairpin structure, 3'terminal base and its stability, etc.;

[0038] 3) Perform gradient PCR amplification and agarose gel electrophoresis EB staining on the synthesized primers for preliminary screening, and determine the opti...

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Abstract

The invention discloses an eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker, the molecular marker is named as SSR1107, and comprises a core sequence (TG) n, an upstream primer and a downstream primer, the upstream primer is shown in SEQ ID NO.1, the downstream primer is shown as SEQ ID NO.2, and n is 7-9. The molecular marker SSR1107 overcomes the defects that a traditional breeding method mainly relies on phenotypic trait identification, and is greatly affected by environmental factors, and the growth trait breeding is difficult and inefficient. Through use of the eriocheir-sinensis-growth-trait-related EST-SSR molecular marker, biological parents excellent in growth trait can be fast and accurately screened for river crab breeding, the selection target is clear, and the time and labor are saved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and genetic breeding, and in particular relates to an EST-SSR molecular marker significantly related to the growth traits of Chinese mitten crab, and also relates to the application of the molecular marker in the breeding of Chinese mitten crab. Background technique [0002] Chinese mitten crab (Eriocheir sinensis), commonly known as river crab, belongs to Crustacea, Decapoda and Eriocheir. It is one of the important economic crustaceans in my country. The breeding areas are spread all over my country's Liaoning, Hebei, Tianjin, Shandong, Jiangsu, Zhejiang and other coastal provinces and vast inland areas. According to statistics in 2009, the national river crab farming involves 30 provinces (municipalities and autonomous regions) across the country, with an area of ​​66.7 million hectares, with an output of 530,000 tons and an output value of 28 billion yuan. It has become the industry w...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 孙金生李晶晶耿绪云薛淑霞魏俊利王雪惠董学旺孙妍
Owner 天津市水生动物疫病预防控制中心
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