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Method for identifying authenticity of brassica rapa variety and special SSR (simple sequence repeat) primer combination of method

A pakchoi, authenticity technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problem of insufficient identification efficiency, inability to judge whether it is a pakchoi variety, and there is no ideal molecular identification yet. methods, etc.

Active Publication Date: 2020-06-05
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is very difficult to verify the authenticity of pakchoi
Especially in the era of the outbreak of the seed industry market, various varieties of pakchoi emerge in endlessly, and it is even impossible to judge whether it is a variety of pakchoi
Relying solely on the general DUS method of phenotypic identification to determine its authenticity can no longer meet the identification needs at this stage, not only because of the lack of identification efficiency, but also its accuracy is questionable
[0004] Based on the method of molecular identification, because there is no reliable reference genome of pakchoi, there is no ideal molecular identification method

Method used

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  • Method for identifying authenticity of brassica rapa variety and special SSR (simple sequence repeat) primer combination of method
  • Method for identifying authenticity of brassica rapa variety and special SSR (simple sequence repeat) primer combination of method
  • Method for identifying authenticity of brassica rapa variety and special SSR (simple sequence repeat) primer combination of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Acquisition of SSR Primer Combination for Identifying the Authenticity of Chinese Cabbage Varieties

[0090] 1. The discovery of 23 SSR loci: These loci were based on the reference genome Brassica_rapa V1.5 of the closely related species of Chinese cabbage and Brassica olerecea V2.1 of the reference genome of cabbage combined with the whole genome resequencing data of 40 germplasm resources obtained by data mining.

[0091] 1. Different from other molecular marker identification methods, the method of blindly selecting SSR markers at random, the present invention uses the reference genome of pakchoi, and uses unique, representative, resequencing data of 40 pakchoi varieties (resequencing results Undisclosed), through a specific big data analysis method, the SSR loci of the whole genome are analyzed, and a series of screening conditions such as PIC, MAF, and sequence conservation are screened, and finally a set of SSR loci is obtained. Data dimensionality reduction and ...

Embodiment 2

[0113] This example is the validity test of the SSR primer combination developed in Example 1.

[0114] 229 pakchoi varieties for testing in the present embodiment are common fine varieties or part imported varieties from abroad, and the specific varieties are as follows:

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] 1. Obtaining the genomic DNA of the pakchoi varieties tested

[0122] The leaves of 229 pakchoi varieties for testing were extracted by CTAB method (the true leaves of 30 seeds were mixed, that is, the true leaves of 30 seeds for each variety, referring to: the same variety of 30 different plants) Genomic DNA of mixed true leaves) to obtain the genomic DNA of pakchoi varieties for testing.

[0123] The operation of the above CTAB method is as follows:

[0124] The leaves of the above-mentioned 229 varieties at the seedling stage were picked respectively, and placed in a freeze dryer (CoolSafe 55-4) to dehydrate; Add 800 μL of ...

Embodiment 3

[0141] This embodiment is a method for detecting whether the variety of pakchoi to be tested belongs to the variety of 229 varieties of pakchoi to be tested. The variety of the pakchoi to be tested is unknown, and the variety of the pakchoi to be tested needs to be obtained through the detection method of this embodiment Whether it is one of these 229 varieties.

[0142] 1. Obtaining the genomic DNA of the Chinese cabbage variety to be tested

[0143] The leaves of the pakchoi varieties to be tested were taken from the experimental base of the Vegetable Research Center of Beijing Academy of Agriculture and Forestry Sciences.

[0144] According to the method of step 1 in Example 2, "leaves of the pakchoi variety to be tested" were replaced with "leaves of the pakchoi variety to be tested", and the other steps were all unchanged to obtain the genomic DNA of the pakchoi variety to be tested.

[0145] 2. Configuration of SSR primers and PCR reaction system

[0146] According to ...

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Abstract

The invention belongs to the field of molecular markers and detection thereof and in particular relates to a method for identifying authenticity of a brassica rapa variety and a special SSR (simple sequence repeat) primer combination of the method. An SSR locus for identifying authenticity of a brassica rapa variety is obtained through data digging according to a reference genome Brassica_rapa V1.5 of brassica rapa and a reference genome Brassica olerecea V2.1 of brassica oleracea together with whole genome resequencing data of 40 genetic resources; the SSR primer combination is selected froma first SSR primer pair to a twenty-third SSR primer pair which are respectively applied to PCR (polymerase chain reaction) amplification on a first SSR locus to a twenty-third SSR locus, and are respectively optimally selected as nucleotide sequences which have 85-100% of homology with nucleotide sequences of SEQ ID NO:1-46 in a sequence table. The method can be applied to authenticity identification on the brassica rapa variety for a whole life cycle from seeds, and technical support is provided for protection on genetic resources and new varieties of brassica rapa.

Description

technical field [0001] The invention belongs to the field of molecular markers and detection thereof, and in particular relates to a method for identifying the authenticity of pakchoi varieties and a combination of special SSR primers. Background technique [0002] Chinese cabbage is a Brassica plant of the genus Brassicaceae. It is native to Asia and is cultivated in all parts of China. [0003] The breeding history of pakchoi is relatively long, but its records are few, especially because its appearance is very similar to other leafy vegetables such as rapeseed, and there are many false claims, so the breeding history of pakchoi is difficult to study. At the same time, due to the U-triangle theory in Brassicaceae breeding, intermediate hybridization is possible, so the genetic background of pakchoi is greatly enlarged from the level of a single crop to the level of interspecies. It is very difficult to verify the authenticity of pak choy. Especially in the current era wh...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 温常龙张建杨静静罗江张凤兰赵岫云于拴仓李佩荣
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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