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Radish genome snp-panel and its application

A genome and radish technology, which is applied in application, plant gene improvement, recombinant DNA technology, etc., can solve the problems of inability to meet individualization, large-scale application, and high price of gene chips, so as to promote agricultural development, improve breeding efficiency, shorten The effect of the breeding cycle

Active Publication Date: 2022-07-12
WUHAN ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, technologies that can be used for molecular marker-assisted breeding include SSR / InDel, KASP, and gene chips, etc. Among them, SSR / InDel and KASP have few markers available and the unit price is high, so they are not suitable for genome-wide molecular marker-assisted breeding; the price of gene chips High and the core technology is owned by foreign countries. It cannot be applied on a large scale and cannot meet individual needs. At the same time, there are technical barriers

Method used

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  • Radish genome snp-panel and its application
  • Radish genome snp-panel and its application
  • Radish genome snp-panel and its application

Examples

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Effect test

Embodiment 1

[0030] Example 1 Extraction of radish genomic DNA

[0031] 1. Choose radish tissue, preferably young tissue, including dry seeds, fresh leaves, young ears and old leaves, seedlings or young stem sections;

[0032] 2. Reagent preparation: CTAB buffer, including 2% CTAB, 1.4M NaCl, 100 mM Tris-HCl, 10 mM EDTA, pH=8.0; washing buffer, including 76% ethanol, 10 mM ammonium acetate; TE buffer, including 20 mM Tris -HCl, 1 MEDTA, pH 8.0; ice anhydrous ethanol, pre-stored anhydrous ethanol at -20°C for more than 1 hour.

[0033] 3. Extract DNA from radish tissue using CTAB method: cut the tissue from radish into pieces and grind in liquid nitrogen environment; add preheated CTAB equivalent to the amount of tissue, and quickly place in a 65°C water bath for 30min To 1h, shake once every 5 minutes (preferably a water bath for 1 hour); after 4 ° C, 12000rpm / min centrifugation for 10min, remove the supernatant and add equal volumes of chloroform and isoamyl alcohol (the volume ratio of ...

Embodiment 2

[0034] Example 2 Designing specific SNP-marked amplification primers

[0035] 1. Selection of SNP markers, analysis of whole genome SNP (the version number of the whole genome sequence is R.sativus_genome_V1.1, the website is http: / / brassicadb.org / brad / datasets / pub / Genomes / Raphanus_sativus / ), by pressing Select 1 SNP variation per 1Mb distance to obtain a large number of SNP markers on the whole genome, wherein the number of the large number of SNP markers evenly distributed on the radish genome is 1000 or more;

[0036] 2. Design of amplification primers. After sequence alignment, analysis and splicing, the genomic data is converted into database files, and Primer3 is used for batch design of amplification primers, and the NCBI-ePCR program is used to analyze the SNP-labeled amplification primers one by one. Test, screening can only amplify a single band containing the SNP marker, and the SNP marker is positioned at the chromosomal site where the template is located, and fina...

Embodiment 3

[0041] Example 3 Detection and analysis of individual genotypes

[0042] 1. Multiplex PCR amplification: the present invention relates to the amplification of 305 SNP markers, 305 SNP markers are combined to form a gene Panel, and the 305 pairs of SNP-marked amplification primers are used to target the extracted radish tissue DNA. The amplified region is amplified; the amplified product is digested and then the adapter is added, the fragments are selected after recovery and purification, and the construction of the next-generation sequencing library is finally completed;

[0043] 2. Quality control and quantification: Including using gel electrophoresis, real-time fluorescent quantitative PCR and Agilent 2100 to conduct quality control and quantification of the constructed library, in preparation for on-machine sequencing;

[0044] 3. Complete PE150 sequencing on the Illumina sequencer to obtain sequence data;

[0045] 4. The SNP genotype results can be obtained by analyzing ...

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Abstract

The present invention relates to radish molecular breeding, in particular to radish genome SNP-Panel and its application. The present invention provides a genome-wide molecular marker detection system with high throughput and low cost, which is a radish genome SNP-Panel, including at least one SNP site in Table 1 and an amplification primer for the SNP site, The primer sequences are shown in SEQ ID Nos. 1-610 in Table 1. The present invention also provides the application of the radish genome SNP-Panel. The radish genome SNP-Panel is a high-efficiency breeding technology system, which can realize the simultaneous detection of 305 SNP loci through one amplification reaction. The technical scheme of the invention can realize the process and informationization of radish molecular design breeding, greatly improve the breeding efficiency of radish in my country, shorten the breeding cycle, and promote agricultural development.

Description

technical field [0001] The present invention relates to radish molecular breeding, in particular to radish genome SNP-Panel and its application. Background technique [0002] Radish is an important cruciferous vegetable crop. In recent years, with the rapid development of high-throughput sequencing technology, the genome information of radish has become more and more abundant, which has laid a good foundation for the development of radish molecular design breeding. Molecular design breeding technology will realize the precise improvement of agronomic traits, showing more prominent advantages than other breeding methods, and is an important direction for the development of crop breeding technology in the future. At this stage, the technologies that can be used for molecular marker-assisted breeding include SSR / InDel, KASP and gene chip, etc. Among them, SSR / InDel and KASP have few available markers and high unit price, which are not suitable for molecular marker-assisted bree...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6806C12N15/11A01H1/04
CPCC12Q1/6895C12Q1/6806A01H1/04C12Q2600/156C12Q2600/13C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122Y02A40/81
Inventor 高长斌张雪丽贺从安张毅梅文康
Owner WUHAN ACADEMY OF AGRI SCI
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