DNA barcode preparation method and Phytophthora fragariae DNA barcode

A technology of Phytophthora fragariae and barcode, applied in the field of DNA detection, can solve the problems of no barcode interval, no DNA barcode research and report, no research and report, etc., to achieve the effect of overcoming limitations

Active Publication Date: 2016-07-06
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, looking at the ideas and methods for the preparation of DNA barcodes of animals, plants, microorganisms, etc., most of them are based on common ITS fragments, mitochondrial pigment oxidase gene Ⅰ, ribosomal large subunit, ribosomal small subunit, RNA Enzyme subunit II, translation elongation factor, mitochondrial small subunit operon, β-vascular bundle protein, actin, chitin synthase gene, calmodulin, heat shock protein 90 and other DNA fragments were used as candidate fragments, and their Species discrimination and identificat

Method used

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  • DNA barcode preparation method and Phytophthora fragariae DNA barcode
  • DNA barcode preparation method and Phytophthora fragariae DNA barcode
  • DNA barcode preparation method and Phytophthora fragariae DNA barcode

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Embodiment

[0028] 1. Materials

[0029] The P.fragariae strain used was numbered 309.62 from the Netherlands Fungal Diversity Research Center CBS. Isolated by C.J.Hickman from Scottish fruit, the host is strawberry.

[0030] 2. Method

[0031] 1. Genomic DNA preparation

[0032] Genomic DNA was extracted using QIAGEN plant and fungal genome preparation kits, and the operation method was as follows:

[0033] (1) Scrape the mycelia from the plate culture medium, avoid scraping the culture medium as much as possible, and grind with liquid nitrogen;

[0034] (2) After the liquid nitrogen grinding is completed, transfer the ground powder to a 1.5mL centrifuge tube, add 400μL BufferAP1 and 4μL RNaseA, vortex, mix well, put in a 65℃ water bath for 10min, and invert 2-3 times during the period;

[0035] (3) Take out the centrifuge tube, directly add 130 μL BufferAP2, shake and mix, and incubate on ice for 5 minutes;

[0036] (4) After culturing on ice, centrifuge, take the supernatant, and ...

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Abstract

The invention discloses a DNA barcode preparation method and a Phytophthora fragariae DNA barcode. The preparation method of the application comprises: sequencing target strain genome DNA by using high-throughput sequencing technology, subjecting sequencing results to bioinformatics analysis, analyzing an evolutional relation of gene fragments in the genome DNA by using a phylogenetic tree to obtain a target strain greater than or equal to 95% and less than 100% in similarity to strains of same genus, and subjecting single copy gene fragments not higher than 1000 bp to genetic distance analysis to obtain the target strain DNA barcode capable of genuinely reflecting genetic relations. The preparation method of the application uses the high-throughput sequencing technology to comprehensively analyze genome DNA and uses the phylogenetic tree and genetic distance analysis to obtain the DNA barcode; a single and effective way to prepare the DNA barcode is provided; the obtained DNA barcode is able to most effectively characterize target strains.

Description

technical field [0001] The application relates to the field of DNA detection, in particular to a method for preparing a DNA barcode and the DNA barcode of Phytophthora fragariae. Background technique [0002] Phytophthora contains more than 100 species and is the most important plant pathogenic fungus. Phytophthorafragariaevar.fragariae (hereinafter abbreviated as P.fragariae) is a pathogen that causes red heart disease in plants of the genus Fragaria. Its hosts include plants of the genus Fragaria and some plants of the genus Rubus, such as loganberries and blackberries. The symptoms caused There are short plants, shrunken leaves and petioles, red hearts in rhizomes, and even the death of the whole plant. Zoospores are the main route by which they infect plants. Not airborne, but spores can survive long in soil and plant material, and spread and spread through drainage and irrigation. Once infected, it can spread to the entire orchard within a few years, seriously affect...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/6895
Inventor 高瑞芳章桂明程颖慧汪莹王颖
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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