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Method for authenticating cucumber variety authenticity and SSR primer group special for method

A primer set, cucumber technology, applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., can solve the problems of simple SSR source, peak slicing, and inability to adapt to automation, etc.

Active Publication Date: 2018-05-04
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current traditional cucumber SSR markers do not refer to the genome variation group information, and there are untruths or variations; in addition, due to the limitation of the SSR detection method, it is easy to lead to untrue, false positive, and false negative results, which are specifically manifested in the following problems: 1) The source of SSR is simple, lacking the support of genome and genetic variation group, poor polymorphism, two-base repeat SSR is easy to produce peaks; 2) There are other SSRs and Indel / SNP interference in the amplification area and motif area of ​​SSR detection, resulting in amplified fragments The size does not change (false negative) or changes; 3) The traditional SSR detection method is to determine the length of the microsatellite marker by gel electrophoresis, which needs to be compared with reference species, which increases the workload of detection and lacks reliable stability of the results and reproducibility; 4) can not meet the needs of automation, high-throughput, low-cost detection

Method used

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  • Method for authenticating cucumber variety authenticity and SSR primer group special for method
  • Method for authenticating cucumber variety authenticity and SSR primer group special for method
  • Method for authenticating cucumber variety authenticity and SSR primer group special for method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1, the acquisition of the SSR primer set used to identify the authenticity of cucumber varieties

[0133] Based on the resequencing data of 49 cucumber representative resources, the present invention develops an SSR primer set suitable for high-throughput sequencing for identifying the authenticity of cucumber varieties. These 49 cucumber resources are rich in types, covering North China type (7), India type (18), Japan type (3), South China type (2), European fruit type (5), American processing type (4 ), Xishuangbanna type (5 copies) and intermediate type (5 copies), which basically included the main ecological types and agronomic traits of cucumber, reflected germplasm representation as much as possible, and had high genetic diversity.

[0134] Specifically, the SSR screening criteria are as follows: no SNP and InDel in the motif region; MAF>0.3 and evenly distributed on the genome; the flanking sequence (200bp) connected to the motif region has no poly re...

Embodiment 2

[0138] The validity check of the SSR primer set that embodiment 2, embodiment 1 develop

[0139] The basic information of the 165 tested cucumber varieties in this example is shown in Table 2. The 165 tested cucumber varieties are all good varieties commonly used in production or some imported varieties from abroad.

[0140] Table 2. Information of 165 cucumber varieties

[0141]

[0142]

[0143] 1. Obtaining the genomic DNA of the tested cucumber varieties

[0144] Genomic DNA of leaves (cotyledons of 5 seeds mixed) of 165 tested cucumber varieties were extracted by CTAB method to obtain genomic DNA of tested cucumber varieties. The quality and concentration of the genomic DNA of the cucumber varieties to be tested must meet the requirements of PCR, and the standard of compliance is: agarose electrophoresis shows a single DNA band without obvious dispersion; the UV spectrophotometer Nanodrop2000 (Thermo) detects that the A260 / A280 ratio is around 1.8 , the ratio of A...

Embodiment 3

[0185] Embodiment 3, the method for detecting whether the cucumber variety to be tested belongs to the variety in 165 cucumber varieties for testing

[0186] 1. Obtaining the genomic DNA of the cucumber variety to be tested

[0187] The leaves of the cucumber varieties to be tested were taken from the experimental base of the Vegetable Research Center of Beijing Academy of Agriculture and Forestry Sciences.

[0188] According to the method of step 1 in Example 2, "the leaves of the cucumber variety to be tested" were replaced with "the leaves of the cucumber variety to be tested", and the other steps were all unchanged to obtain the genomic DNA of the cucumber variety to be tested.

[0189] 2. Preparation of PanelMix primer mixture

[0190] Same as step 2 in Example 2.

[0191] 3. Preparation of sequencing library

[0192] According to the method of step 3 in Example 2, replace "the genomic DNA of the cucumber variety to be tested" with "the genomic DNA of the cucumber vari...

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Abstract

The invention discloses a method for authenticating cucumber variety authenticity and an SSR primer group special for the method. The SSR primer group provided by the invention consists of 32 primers;the nucleotide sequences of the 32 primers are as shown in sequence 1 to sequence 3 in a sequence table. The SSR primer group provided by the invention has the advantages of high polymorphism, high repeatability, stable and reliable marking, convenience in statistics and the like. According to the method for authenticating the cucumber variety authenticity and the SSR primer group special for themethod, a DNA fingerprint database for authenticating the cucumber variety authenticity on the basis of high-throughput sequencing is also established and can be used for performing early authenticating on a cucumber variety in a seed or young seeding stage, so that the authenticity of the variety is ensured, the rights and interests of a producer and a breeder are practically protected, and technical support is provided for cucumber germplasm resource and new variety protection. Therefore, the method for authenticating the cucumber variety authenticity and the SSR primer group special for the method have a broad application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying the authenticity of cucumber varieties and a special SSR primer set thereof. Background technique [0002] my country is the country with the largest cucumber cultivation area (about 1.396 million hectares) and the highest total output (accounting for about 70% of global output) in the world. Since the reform and opening up, the cucumber industry has developed rapidly, and more than 300 varieties have passed the national examination and registration. Due to the small and scattered cucumber breeding enterprises, the variety management has not been effectively followed up, and the variety approval among various provinces and cities is not coordinated with each other. In addition, the management of the production base of fine seeds is chaotic, the phenomenon of stolen breeding and arbitrage of varieties is rampant, the phenomenon of homonym is serious, the qual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 温常龙张建杨静静张峰毛爱军许勇
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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