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KASP primers for detecting cucumber bacterial angular leaf spot gene of and application thereof

A technology for bacterial and angular spot disease, which is applied in the fields of molecular biology and crop breeding, can solve the problems of inability to determine the resistance of cucumber bacterial angular spot, long time-consuming, low accuracy of fluorescence detection, etc., to achieve accurate anti-corrosion Disease identification, speed up the breeding process, and improve the effect of breeding efficiency

Pending Publication Date: 2019-09-17
河北省农林科学院经济作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first problem in the above patent literature is that it provides molecular markers for the detection of bacterial angular leaf spot of cucumber, wherein the detection is whether the sample carries bacterial angular leaf spot of cucumber, or whether the plant is infected by bacterial angular leaf spot
It is not possible to detect whether the material to be tested carries the resistance gene of bacterial angular spot of cucumber, nor can it determine the resistance of the material to be tested to bacterial angular spot of cucumber
The second is to use conventional PCR for detection, which requires three rounds of PCR amplification, which is time-consuming and costly; the requirements for DNA samples are high, and the CTAB method needs to be used to extract whole-genome DNA for PCR amplification, which is complicated and takes a long time ; Then gel electrophoresis is carried out to judge the results, and its accuracy is relatively low compared with the fluorescence detection of KASP
The problems in the above-mentioned patent documents are that the patent detects whether the sample carries the bacterial angular leaf spot pathogen of cucumber, or detects whether the plant is infected by the bacterial angular leaf spot pathogen
It is not possible to detect whether the material to be tested carries the resistance gene to bacterial angular spot of cucumber, nor can it determine the resistance of the material to be tested to bacterial angular spot of cucumber; The sample requirements are high, and the whole genome DNA needs to be extracted for PCR amplification, and then gel electrophoresis is used to judge the results. Compared with the fluorescence detection of KASP, the accuracy is relatively low; the third is the infection quantity of bacterial angular leaf spot of cucumber There are certain requirements and limitations

Method used

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  • KASP primers for detecting cucumber bacterial angular leaf spot gene of and application thereof
  • KASP primers for detecting cucumber bacterial angular leaf spot gene of and application thereof
  • KASP primers for detecting cucumber bacterial angular leaf spot gene of and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] KASP mark development

[0077] Comparative analysis of the sequences of the two alleles of CsPSL and CsPSL323. Among them, CsPSL is a non-resistant allele, and CsPSL323 is a resistant allele, and there is a SNP locus at the position of 323bp between the two.

[0078] The SNP sites that can be used for the development of KASP markers must meet the following requirements: CsPSL and CsPSL323 have different genotypes; there are no other SNP sites nearby, and they are located in non-SNP dense regions; avoid continuous AT, high GC content and other complex sequences.

[0079] Therefore, the A / G locus at position 323 is the only selection target; the genotype of CsPSL in this locus is A, and the genotype of CsPSL323 is G.

[0080] Design primers for the above-mentioned sites, and their sequence is (wherein the underlined sequence is the added tag sequence):

[0081] No. 323 A / G:

[0082] Forward primer 1: 5’-GAAGGTGACCAAGTTCATGCTCAAGGATGGTATAATTGGCTTCA-3’;

[0083] Forward primer 2: 5'-G...

Embodiment 2

[0087] Amplification of molecular markers

[0088] The materials used in this example are the known anti-bacterial angular leaf spot material GY14 of cucumber and the material 9930 susceptible to bacterial angular leaf spot of cucumber.

[0089] The genomic DNA from cucumber leaves was extracted by alkaline boiling method, and the concentration of the extracted DNA solution was 10-50ng / μl and stored at -20°C.

[0090] Use KASP-PARMS kit (Jing peptide biological company) to configure the PCR reaction system, the total reaction volume is 10μl, including: 2X PARMS PCR Mix, 5μl; DNA extraction solution (10-50ng / μl), 1μl; forward primer CsPSL- F1, 0.15μl (10pmol / μl); forward primer CsPSL-F2, 0.15μl (10pmol / μl); reverse primer CsPSL-R, 0.4μl (10pmol / μl); and ddH2O, 4.3μl. Set three technical repetitions.

[0091] The PCR reaction program is: 94°C for 15 minutes; 94°C for 20 seconds, 65°C (decreased by 0.8°C per cycle) for 1 minute, 10 cycles; 94°C for 20 seconds, 57°C for 1 minute, 28 cycl...

Embodiment 3

[0093] Detection and analysis of amplified products

[0094] Use Tecan Infinite M200 multifunctional microplate reader to genotype PCR products, use SNPDecoder online data analysis software (http: / / www.snpway.com / snpdecoder / ) to analyze the data, and set the FAM fluorescence signal close to On the horizontal axis, the HEX fluorescence signal is close to the vertical axis.

[0095] See the results of marker typing figure 1 . In the figure, the horizontal and vertical coordinates indicate the signal value; the dot I is the amplified signal of the susceptible material 9930 (only FAM fluorescent signal is detected); the dot III is the amplified signal of the disease-resistant material GY14 (only the HEX is detected) Fluorescence signal); The dot at Ⅱ is the hybrid disease-resistant material GY14-9930-F1, and HEX fluorescence and FAM fluorescence are detected at the same time; the gray dot near the origin represents the amplification signal of the negative control (no DNA sample added)...

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Abstract

The invention relates to the fields of molecular biology and crop breeding technology, and in particular to KASP primers for detecting a cucumber bacterial angular leaf spot gene and an application thereof. The cucumber bacterial angular leaf spot resistant gene CsPSL has an SNP locus at the position of 323. Based on the SNP locus, KASP primers and a kit comprising the KASP primers are developed. The KASP primers of the present invention and the kit containing the KASP primers can be used for identifying whether the cucumber contains the cucumber bacterial leaf spot resistant gene, and distinguishing the cucumbers against the cucumber bacterial leaf spot and the cucumbers susceptible to the cucumber bacterial leaf spot. Molecular markers provided by the present application can quickly and accurately identify the disease resistance of cucumber plants in a seedling stage, thereby improving breeding efficiency, saving breeding cost and speeding up the breeding process.

Description

Technical field [0001] The invention relates to the technical fields of molecular biology and crop breeding, in particular to a KASP primer for detecting cucumber bacterial angular spot disease gene and its application. Background technique [0002] Cucumber (Cucumis sativus L.) is the third largest vegetable crop in the world, and its planting area in China ranks first in the world. Cucumber Angular leaf spot (ALS) is one of the most devastating diseases in cucumber production worldwide. With the growing expansion of greenhouse planting areas in my country, cucumber bacterial angular leaf spot has become one of the most serious diseases in greenhouse cucumber production, which seriously affects the quality and yield of fruits and causes serious economic losses. Therefore, it is necessary to select disease-resistant varieties. It seems very important. [0003] Bacterial keratoderma is a leaf disease caused by Pseudom onassyringae pv. Lachrymans (psl), but it can also cause harm to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 吴志明康忱李亚栋朱金城田哲娟高振华齐连芬
Owner 河北省农林科学院经济作物研究所
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