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High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof

A tomato leaf mold and molecular marker technology, applied in the field of agricultural biology, can solve problems such as low detection efficiency, high cost, and cumbersome experimental procedures, and achieve the effects of reducing human error, high analysis throughput, and saving time and labor costs

Active Publication Date: 2016-06-01
SHANDONG SHOUGUANG VEGETABLE SEED IND GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a low-cost, fast, efficient, and high-accuracy identification of tomato leaf mold resistance gene Cf for the deficiencies and defects in the prior art that require enzymatic digestion, high cost, cumbersome experimental procedures, and low detection efficiency. High-throughput molecular labeling of -5 and its labeling method and application

Method used

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  • High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof
  • High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof
  • High-flux molecular marker for identifying tomato leaf mold resistance, and marking method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design of high-throughput molecular markers for identification of tomato leaf mold resistance gene Cf-5

[0052] (1) More than 20 copies of tomato materials with Cf-5 resistance and tomato materials without Cf-5 resistance were taken from different sources, and their whole genome DNA was extracted by CTAB method:

[0053] 1) Freeze-dry the tomato leaves, after grinding, take 0.6g of the sample and add it to a 1.5mL centrifuge tube containing 1mL of preheated CTAB, put it in a water bath at 65°C for 30min, and invert it 3-5 times during the process;

[0054] 2) Add 400 μL of chloroform: isoamyl alcohol mixture (volume ratio 24:1), mix upside down, and centrifuge at 12000 rpm for 15 min;

[0055] 3) Take the supernatant, add an equal volume of isopropanol, mix it upside down, and place it at -20°C for 30 minutes;

[0056] 4) Centrifuge at 12000rpm for 10min, discard the supernatant, and wash once with 70% alcohol;

[0057] 5) to dry, add 60μLddH 2 O was diss...

Embodiment 2

[0081] Example 2: Establishment of a method for detecting tomato Cf-5 gene using high-throughput molecular markers

[0082] 1. Genomic DNA extraction

[0083] Referring to Example 1 Genomic DNA Extraction Method.

[0084] 2. PCR amplification

[0085] Different tomato genomic DNAs are used as templates, and the molecular markers of the present invention are used for PCR amplification to obtain PCR amplification products.

[0086] Genotyping PCR reaction system:

[0087] 96-well plate: 10ng genomic DNA, 5ulKASPV4.02×MasterMix, 0.14ulKASP72×assaymix, add ddH 2 0 to 10ul.

[0088] 384-well plate: 5ngDNA, 2.5ulKASPV4.02×MasterMix, 0.07ulKASP72×assaymix, add ddH 2 0 to 5ul.

[0089] 1536-well plate: 5ngDNA, 2.5ulKASPV4.02×MasterMix, 0.07ulKASP72×assaymix, add ddH 2 0 to 5ul.

[0090] Among them, KASPV4.02×MasterMix is ​​a product of LGC, the catalog number of KASPV4.02×MasterMix for 96 / 384-well plates is KBS-1016-002; the catalog number of KASPV4.02×MasterMix for 1536-well ...

Embodiment 3

[0102] Example 3: Application of high-throughput molecular markers of tomato Cf-5 gene in tomato variety breeding

[0103] 1) Using high-throughput molecular markers of the tomato Cf-5 gene to detect tomato breeding materials, including 200 F3 generation materials, 200 F4 generation breeding materials, 200 F5 generation breeding materials and 200 F6 generation breeding materials, specifically Method with reference to embodiment 2.

[0104] 2) Carry out artificial inoculation identification of tomato leaf mold on individual tomato plants detected by molecular markers. In the 4-5 leaf stage, spray the tomato leaf surface with a concentration of 7-8 spore suspensions (under a field of view of 200×) for inoculation, observe and record the incidence of tomato plants after 2 weeks.

[0105] 3) By comparing and analyzing the results of high-throughput molecular marker analysis and artificial inoculation results, it was found that the coincidence rate of genotype and phenotype was as...

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Abstract

The invention discloses a high-flux molecular marker for identifying tomato leaf mold resistance, and a marking method and application thereof. According to the molecular marker, a primer Cf-5-Al lele-F1, a primer Cf-5-Al lele-F2 and a primer Cf-5-R are designed according to the key specific sites of the target gene, and a primer tail end base is specifically matched with the target gene to carry out genotyping. A PCR (polymerase chain reaction)-based SNP Line platform is a high-flux molecular marker system, and thus, is capable of implementing the full-automatic operation procedure and lowering the human errors. The molecular marker has high analysis flux, can complete 0.5 million SNP genotyping every day, and is suitable for simultaneously detecting abundant samples. The high-detection-flux KASP molecular marker designed on the basis of tomato-leaf-mold-resistant gene Cf-5 is used for high-flux screening and identification of tomato-leaf-mold-resistant plants, can greatly save the time and labor cost, enhances the breeding efficiency of molecular marker assisted selection, has important meanings for culturing leaf-mold-resistant tomato leading varieties and effectively controlling hazards of tomato leaf mold, and is important breeding method innovation and technical innovation.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and relates to a high-throughput molecular marker for identifying tomato leaf mold resistance, a marker method and application thereof. Background technique [0002] Tomato is one of the important vegetable crops in my country, and occupies a very important position in the vegetable production in protected areas. Tomato leaf mold (Cladosporium fulvum Cooke) is one of the worldwide diseases that threaten tomato production in protected areas. It seriously affects the quality of tomatoes, leads to tomato yield reduction, and brings huge losses to tomato production in protected areas. Tomato leaf mold was first reported in the UK in 1883. Due to the rapid spread and the outbreak of disasters in a short period of time, scientists soon attached great importance to it. After tomato leaf mold occurred in Taiwan in the 1930s, it occurred successively in mainland China. With the expansion of prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 程琳张生国家进陈福东张晓燕董甜李晓杰刘岳王如芳董静于彩玉
Owner SHANDONG SHOUGUANG VEGETABLE SEED IND GRP CO LTD
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