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KASP-based soybean core SNP markers and application thereof

A soybean and labeling technology, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of different seeds, high chip labeling density, high detection cost, etc.

Inactive Publication Date: 2021-05-18
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These high-throughput chips are gradually used in the identification of soybean germplasm resources, but the density of such chip markers is relatively high, and the common problem is that a specific detection platform is required, and the detection cost is high, and the detection flexibility is relatively poor
Not only do there exist counterfeit and shoddy varieties with the same name but different seeds, and the same seeds but different names in the seed market, it is also found in the annual soybean regional tests that the genotype and phenotype are the same, but the lines from different breeders participate in the test, causing national Waste of human and material resources

Method used

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  • KASP-based soybean core SNP markers and application thereof
  • KASP-based soybean core SNP markers and application thereof
  • KASP-based soybean core SNP markers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Screening of soybean core SNP markers

[0025] Through the detection results of 5,000 soybean resources by the "Illumina Zhongdouxin No. 1" chip, according to the principle of minimum allelic variation (MAF) ≥ 0.4, select SNP sites on different soybean chromosomes to ensure that all materials have a similarity <95% 65 high-resolution SNP sites were screened (Table 1), and KASP detection markers were developed and verified with 280 soybean resources.

[0026] Table 1 65 core SNP markers of soybean resources

[0027]

[0028]

[0029] The above SNP physical locations were determined based on the reference genome Wm82.a2.v1 (Phytozome, https: / / phytozome.jgi.doe.gov / pz / portal.html).

Embodiment 2

[0030] Example 2 A method for identifying 280 soybean resources using 65 soybean core SNP markers

[0031] (1) DNA preparation

[0032] Genomic DNA was extracted from 280 soybean leaves or soybean flour by CTAB method.

[0033] (2) Design and synthesis of KASP primers

[0034] The primer working solution was prepared by mixing three primers (Table 2), and the concentration of each primer was: AlleleFAM primer concentration 12 μM, AlleleHEX primer concentration 12 μM and universal primer (Common) concentration 30 μM. The 5' end of the AlleleFAM primer is labeled with FAM, and the 5' end of the AlleleHEX primer is labeled with HEX.

[0035] Table 2 KASP primer sequence (5'-3')

[0036]

[0037]

[0038]

[0039]

[0040] (3) PCR amplification

[0041] PCR system: PCR amplification-related reagents were added to the Array Tape, and the reaction system was shown in Table 3.

[0042] Table 3 PCR amplification system based on Douglas Array tape platform

[0043] ...

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Abstract

The invention discloses KASP-based soybean core SNP markers and application thereof, and belongs to the technical field of molecular detection. The KASP-based SNP markers comprise 65 core SNP markers for identifying soybean resources. Compared with the conventional high-throughput SNP chip detection, the KASP-based soybean core SNP markers have the advantages that only a sequential method is needed for analysis during variety weight determination or purity analysis, and other sites do not need to be detected when different sites required by weight determination are reached; and therefore, the KASP markers developed by the invention have detection flexibility in the aspects of samples, the number of sites and the like, and are suitable for rapid typing of a large number of samples.

Description

technical field [0001] The invention relates to the technical field of molecular detection, in particular to KASP-based soybean core SNP markers and applications thereof. Background technique [0002] my country is the country of origin of soybean and has the most abundant soybean germplasm resources. At present, there are more than 30,000 cataloged resources, including 27,827 cultivated soybeans, 6,708 wild soybeans, and 3,218 imported germplasms. The applicant's project team is responsible for the collection, evaluation and utilization research of national soybean germplasm resources. It has started to study the sampling method of core germplasm 7-10 years earlier than other countries, and carried out the evaluation of molecular marker detection (SSR markers) to construct core species. quality. The development and utilization of SNP chips have also been reported internationally in recent years. The Soybean Genomics and Improvement Laboratory of the US Department of Agricu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 邱丽娟关荣霞郭潇阳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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