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Radish genome SNP-Panel and application thereof

A genome and radish technology, applied in the fields of application, plant genetic improvement, recombinant DNA technology, etc., can solve the problems of inability to large-scale application, inability to meet individualization, and high price of gene chips, to promote agricultural development, improve breeding efficiency, shorten the Effects of Breeding Cycles

Active Publication Date: 2021-12-03
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

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  • Radish genome SNP-Panel and application thereof
  • Radish genome SNP-Panel and application thereof
  • Radish genome SNP-Panel and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Extraction of Radish Genomic DNA

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

[0032] 2. Reagent preparation: CTAB buffer, including 2% CTAB, 1.4M NaCl, 100mM Tris-HCl, 10mM EDTA, pH=8.0; Washing buffer, including 76% ethanol, 10mM ammonium acetate; TE buffer, including 20mM Tris -HCl, lmMEDTA, pH8.0; ice absolute ethanol, pre-store absolute ethanol at -20°C for more than 1 hour.

[0033] 3. Use the CTAB method to extract DNA from radish tissue: cut the tissue from radish into pieces, and grind it in a liquid nitrogen environment; add preheated CTAB equivalent to the amount of the tissue, and quickly place it in a water bath at 65°C for 30 minutes Shake once every 5 minutes (preferably in a water bath for 1 hour); centrifuge at 4°C and 12000rpm / min for 10min, remove the supernatant and add an equal volume of chloroform and isoamyl alcohol (the volume ratio o...

Embodiment 2

[0034] Example 2 Design of amplification primers for specific SNP markers

[0035] 1. The selection of SNP markers, analyze the 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 Selecting one SNP variation per 1Mb distance to obtain a large number of SNP markers on the whole genome, wherein the number of a large number of SNP markers evenly distributed on the radish genome is 1000 or more;

[0036] 2. Amplification primer design, after sequence comparison, analysis and splicing, after converting the genome data into a database file, use Primer3 to design the amplification primers in batches, and use the NCBI-ePCR program to process the SNP-marked amplification primers one by one Test, screening can only amplify a single band containing the SNP marker, and locate the SNP marker at the chromosomal site where the template is located, and finally...

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. Using the amplification primers of the 305 pairs of SNP markers, the extracted radish tissue DNA target The region is amplified; the amplified product is digested and an adapter is added, the fragment is selected after recovery and purification, and the construction of the next-generation sequencing library is finally completed;

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

[0044] 3. Complete PE150 sequencing on an illumina sequencer to obtain sequence data;

[0045] 4. Analyze the sequence data obtained by sequencing to obtain the SNP genotype result. Man...

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Abstract

The invention relates to radish molecular breeding, in particular to a radish genome SNP-Panel and application thereof. The invention provides a molecular marker detection system which is high in flux and low in cost and covers a whole genome; the molecular marker detection system is the radish genome SNP-Panel and comprises at least one SNP site in a table 1 and an amplification primer of the SNP site; and the primer sequence is shown as SEQ ID No.1-610 in the table 1. The invention also provides an application of the radish genome SNP-Panel. The radish genome SNP-Panel is an efficient breeding technology system, and simultaneous detection of 305 SNP loci can be realized through one-time amplification reaction. According to the technical scheme, flow and informatization of radish molecular design breeding can be achieved, the radish breeding efficiency in China is greatly improved, the breeding period is shortened, and agricultural development is promoted.

Description

technical field [0001] The invention relates to radish molecular breeding, in particular to radish genome SNP-Panel and application thereof. Background technique [0002] Radish is an important cruciferous vegetable crop. With the rapid development of high-throughput sequencing technology in recent years, the genome information of radish has become more and more abundant, which has laid a good foundation for molecular design and breeding of radish. Molecular design breeding technology will achieve precise improvement of agronomic traits, showing more prominent advantages than other breeding methods, and will be an important direction for the development of crop breeding technology in the future. 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 bree...

Claims

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Application Information

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Patent Type & Authority Applications(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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