Aristichthys nobilis microsatellite marker primer and aristichthys nobilis marker release effect evaluation method

A microsatellite marker and microsatellite technology, applied in the field of animal molecular genetics, can solve problems such as rare reports of effects, and achieve the effect of great promotion value

Pending Publication Date: 2021-11-09
武汉中科瑞华生态科技股份有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the effect of releasing bighead carp, and there is an urgent need for an effective method to evaluate the effect of artificial proliferation and releasing of bighead carp

Method used

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  • Aristichthys nobilis microsatellite marker primer and aristichthys nobilis marker release effect evaluation method
  • Aristichthys nobilis microsatellite marker primer and aristichthys nobilis marker release effect evaluation method
  • Aristichthys nobilis microsatellite marker primer and aristichthys nobilis marker release effect evaluation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] DNA Extraction of Bighead Carp

[0039] 1) Preparation for digestion. To clean the tools, take 50-100 mg of samples fixed in absolute ethanol, soak them in deionized water for 20 minutes, and remove the alcohol.

[0040] 2) Digestion. Cut the sample into pieces, put it in a 1.5ml centrifuge tube, add 200 μL of DNA lysate, 5 μL of proteinase K (10 mg / mL), centrifuge slightly, mix it upside down, put it in a 50℃~55℃ water bath for 3~5 hours, until the lysate clarify.

[0041] 3) extraction. Add 200 μL of phenol: chloroform: isoamyl alcohol (25:24:1), shake upside down for 15-20 min, centrifuge at 8000 rpm for 5 min, suck out the supernatant, and place it in a new 1.5 mL centrifuge tube. repeat three times.

[0042] 4) Precipitation. Add 800 μL of dehydrated ethanol pre-cooled at -20°C (if a flocculent precipitate occurs, it is a DNA sample with high purity, which can be picked out for use.), centrifuge at 12,000 rpm for 15 minutes, pour off the supernatant, and add ...

Embodiment 2

[0047] Acquisition of microsatellite primers

[0048]The genome and transcriptome sequences of bighead carp were downloaded from NCBI, microsatellite search software was used to search for microsatellite fragments in the genome and transcriptome, and microsatellite primers were designed using Primer 6 software for the fragments containing microsatellites. For microsatellite primers with primer product fragment length greater than 100bp, primer Tm value less than 61°C, and Tm value difference between upstream and downstream primers within 1°C, select 150 pairs of primers. Finally, it was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for the synthesis of primers.

Embodiment 3

[0050] Microsatellite primer screening

[0051] Five bighead carp DNA templates were selected for preliminary screening of the microsatellite primers synthesized in Example 2, and microsatellite markers capable of stable amplification were selected. PCR reaction program: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing temperature for 30s, extension at 72°C for 30s, 30 cycles; extension at 72°C for 10min; storage at 4°C, PCR reaction system: 10×PCR Buffer 3ul, 2.5 mmol / LdNTP 2ul, 2mmol / L MgCl 2 3ul, 1ul each of upstream and downstream primers, 5U / ul Taq enzyme 0.5ul, DNA template 2ul, ultrapure water 12.5ul. The PCR products were detected by polyacrylamide gel with a volume fraction of 8%, and scanned and stored. If there were many bands, the annealing temperature could be changed for re-amplification. The conventional reagents used in polyacrylamide gel were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

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Abstract

The invention belongs to the field of animal molecular genetics, and relates to an aristichthys nobilis microsatellite marker primer and an aristichthys nobilis marker release effect evaluation method; the evaluation method comprises marking, sample collection, sample genetic archive construction, release group recapture, recaptured individual identification and final feedback of an enhancement and release effect according to the recapture rate; and therefore, the contribution of the aristichthys nobilis enhancement and release to the aristichthys nobilis ecological quantity of a release water area is estimated, so that the aristichthys nobilis enhancement and release effect is determined. According to the method, the proportion of aristichthys nobilis released in the water area can be effectively identified, so that the release effect of the aristichthys nobilis is evaluated, and the method has a huge popularization value.

Description

technical field [0001] The invention belongs to the field of animal molecular genetics, and relates to a bighead carp microsatellite marker primer and a method for evaluating the release effect of the bighead carp marker. Background technique [0002] The intensity of human activities has strongly affected the fish living environment on a global scale, resulting in a sharp decline in the diversity and resources of fish. Breeding and release is to artificially release aquatic biological seedlings or parents into public waters such as oceans, rivers, and lakes to increase the strength of population replenishment. The effect of multiplication and release should be said to be very good, which can be reflected from the following aspects: 1) aquatic biological resources can be replenished and restored through multiplication and release; 2) for some endangered species, the way of multiplication and release can increase their wild The number of populations and the promotion of gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2600/156C12Q2525/151
Inventor 陶敏黄强李红涛汤婷陈仁峰张宏刘长明彭海波杨俊锋李想宋江腾
Owner 武汉中科瑞华生态科技股份有限公司
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