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Development method and breeding application of rice 1K SNP-Panel

A 1ksnp-panel, rice technology, applied in the development method and breeding application field of rice 1KSNP-Panel, can solve the problems of inability to large-scale application, unfavorable breeding safety, high unit price, meet the needs of molecular breeding, promote agricultural development, and speed up The effect of development time

Inactive Publication Date: 2021-06-11
武汉基诺赛克科技有限公司
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003]Current molecular marker-assisted breeding technologies include SSR / InDel, KASP and gene chips. SSR / InDel and KASP have few available markers and high unit price, so they are not suitable for the whole genome molecular marker-assisted breeding; the price of gene chips is high and the core technology is owned by foreign countries, which cannot be applied on a large scale and cannot meet individual needs. At the same time, there are technical barriers. In view of the current international situation, it is not conducive to the safety of domestic breeding

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  • Development method and breeding application of rice 1K SNP-Panel

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Effect test

Embodiment 1

[0025] Embodiment one, the extraction of rice genomic DNA

[0026] 1. Select rice tissues, preferably young tissues, including dry seeds, fresh leaves, young ears and old leaves, seedlings or young stems;

[0027] 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, 1mM EDTA, pH 8.0; ice absolute ethanol, pre-store absolute ethanol at -20°C for more than 1 hour.

[0028] 2. Use the CTAB method to extract DNA from rice tissue: chop the tissue from rice 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 until 1 hour, preferably in a water bath for 1 hour; 4°C, centrifuge at 12,000 rpm / min for 10 minutes, remove the supernatant and add an equal volume of chloroform:isoamyl alcohol (24:1), mix w...

Embodiment 2

[0029] In the second embodiment, amplification primers for specific SNP markers are designed.

[0030] 1. Selection of SNP markers, based on the reported rice genome resequencing data and the reported cloned rice important functional genes, a large number of SNP markers evenly distributed on the rice genome were discovered through methods including clustering and splicing, Wherein the number of a large number of SNP markers evenly distributed on the rice genome is 1000 or more than 1000;

[0031] 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 obtain the amplification primer pair...

Embodiment 3

[0036] Embodiment 3, detection and analysis of individual genotype.

[0037] 1. Multiplex PCR amplification, the present invention relates to the amplification of 1253 SNP markers, 1253 SNP markers are combined to form a gene Panel, using the 1253 pairs of SNP marker amplification primers to target the extracted rice tissue DNA 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;

[0038] 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;

[0039] 3. Complete PE150 sequencing on an illumina sequencer to obtain sequence data.

[0040]4. Use "GKPanel" bioinformatics software for analysis, among which "GKPanel" is a self-develo...

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Abstract

The invention relates to the gene detection technology applied to the field of molecular breeding, in particular to a development method and breeding application of rice 1K SNP-Panel. The gene detection technology comprises 1253 SNP markers which uniformly cover the whole genome of the rice, 10000 samples can be detected at the same time at most, a pair of corresponding special primers is provided for each SNP marker, the primers are subjected to high-throughput detection through multiple PCR and a high-throughput sequencing technology to obtain DNA sequence data, and the DNA sequence data are compared with the single nucleotide polymorphism (SNP) markers. Through independently developed special software 'GKPanel', specified data analysis is automatically completed, and corresponding genotype data is given. Gene detection and analysis requirements in the field of molecular breeding can be met by utilizing the technology, the detection coverage is wide, the analysis speed is high, the detection cost is low, the rice 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 a gene detection technology applied in the field of molecular breeding, in particular to the development method and breeding application of rice 1K SNP-Panel. Background technique [0002] The "Molecular Design and Breeding of Main Economic Crops" project is the first batch of key projects launched by the National Key R&D Program "Seven Major Crop Breeding"; molecular design breeding technology will achieve precise improvement of agronomic traits, which is more prominent than other breeding methods The superiority is the future direction of crop breeding technology development. Rice SNP-Panel is a high-efficiency breeding technology system, which can realize the flow and informatization of rice molecular design breeding, greatly improve the efficiency of rice breeding in my country, shorten the breeding cycle, and promote agricultural development; through the accumulation of rice SNP-Panel technology development, relevant The t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895A01H1/04
CPCC12Q1/6895A01H1/04C12Q2600/156C12Q2600/13
Inventor 张毅王博李娟娟王娟张倩柳依
Owner 武汉基诺赛克科技有限公司
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