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Combination and application of mnp marker primers for the identification of rapeseed varieties and their substantive derivatives

A combination of primers and variety identification technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of damaging the rights and interests of breeders and rapeseed growers, disrupting the rapeseed variety market, etc.

Active Publication Date: 2021-07-13
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, with the loosening of conditions for the identification of rapeseed varieties and the rapid development of the market, rapeseed varieties have blown out, but there will be many licensed varieties on the market, seriously disrupting the rapeseed variety market, and ultimately damaging the rights and interests of both breeders and rapeseed growers , in view of the above problems, the present invention provides a set of MNP primer sequences related to the identification of rapeseed varieties and derivatives thereof, using this set of primers can carry out efficient and accurate identification of rapeseed varieties and derivatives thereof, purifying the rapeseed variety market

Method used

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  • Combination and application of mnp marker primers for the identification of rapeseed varieties and their substantive derivatives
  • Combination and application of mnp marker primers for the identification of rapeseed varieties and their substantive derivatives
  • Combination and application of mnp marker primers for the identification of rapeseed varieties and their substantive derivatives

Examples

Experimental program
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Effect test

Embodiment 1

[0019] MNP-marked primer combinations for identification of Brassica napus varieties and their substantial derivatives are obtained in the following manner:

[0020] The applicant has passed the analysis of 19 DH pure lines of Brassica napus (respectively: rich oil selection, R18, gys1, gys3, gys4, Zhong4, DP-10, Z50, T2369 selection, J520 ​​selection, Dadi 95, Chu79 selection, 17HL selection) , 5-9994-5, L3, Ohui, Ohui1, anti-tumor root OR and OR anti-cracking angle) (Liu Chengming et al., 1994; Tang Shuangxi et al., 2012; Wang Jinxiong et al., 2019), genome resequencing and biological Informatics analysis, 511 pairs of MNP marker primers that are evenly distributed in the rape genome were selected. In the marker screening, the high polymorphism in the amplification region, the conservation of the primer region, the selection of single-copy regions in the amplification region, and the deletion in different varieties were observed. General principles for primer screening with ...

Embodiment 2

[0022] Effect verification of MNP-labeled primer set:

[0023] (1) Sample purity requirements

[0024] The seed purity of all crops to be tested is not less than 95%. The requirements of this standard are consistent with the national standards for the purity of field seeds, so as to avoid the influence of hybrid seeds on the typing results.

[0025] (2) Experimental operation

[0026] In this standard, the number of multiplex amplification cycles is less than 20, and it is in a linear growth phase. The detection of amplified linear products by next-generation high-throughput sequencing can achieve relative quantification between alleles. Using quantitative results, aerosol contamination of the laboratory can be ruled out. Therefore, this standard is not very strict on the prevention of aerosol pollution. It only requires experimental partitions, one-way flow, dedicated equipment and ventilation, and these requirements can be met in ordinary laboratories, reducing the requir...

Embodiment 3

[0084] Application of MNP-tagged primer combinations for Brassica napus variety identification:

[0085] The two closely related lines, Z50 and gys4, were identified and analyzed for substantial derived lines. Z50 was the original parent, while the gys4 line had only 16% marker discrimination in terms of genetic distance. From the breeding process and field phenotypes From the point of view, they only have subtle phenotypic differences such as oleic acid content and flowering period. It is feasible to use this set of MNP markers to identify the substantial derivation between materials and its breeding material source and breeding process.

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Abstract

The invention relates to the field of molecular identification of rapeseed varieties, and discloses a nucleotide sequence related to the identification of rapeseed varieties and the determination of substantially derived varieties and its application. Through genome resequencing and bioinformatics analysis of 19 DH pure lines of Brassica napus, the applicant selected 511 pairs of MNP-labeled primers evenly distributed in the rapeseed genome, and performed multiple amplification, second-generation high-throughput sequencing and Data analysis has realized the detection of 19*511=9709 markers in one experiment, and an average of 484 (95%) MNP markers can be detected from each variety, the number of detected markers far exceeds the existing SSR markers, and the detection of The rate is high. Five of the samples were independently tested three times by different people, instruments and time, and the logarithm of the difference in the genotype of the MNP markers of the samples was 0, and the accuracy was 100%. A set of rapeseed MNP marker primer combinations developed by us can effectively distinguish rapeseed varieties and their essential derivatives.

Description

technical field [0001] The invention relates to the field of molecular identification of rapeseed varieties, in particular to the combination and application of MNP marker primers for identification of rapeseed varieties and their substantially derived varieties. Background technique [0002] Rape belongs to the Brassica family, and is an important oilseed economic crop in China. my country is a major producer of rapeseed, with an average annual planting area and total output accounting for about 1 / 3 of the world's. Rape is a cross-pollinated crop with high heterozygosity. Moreover, it is a tetraploid crop with a complex genome. Rapeseed hybrids have the advantages of high yield, strong stress resistance and wide adaptability, and are favored by seed companies and farmers. With the implementation of the "registration system" reform for rape and other crops, the rapid development of hybrid rape varieties and the continuous advancement of breeding technology, the emergence o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 刘佳胡琼彭海李甜甜梅德圣汪文祥成洪涛李超付丽
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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