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Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker

A technique for restoring genes and molecular markers, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., and can solve the problems of shortening the breeding process, long breeding cycle, and limited distribution of fertility restoration genes and other problems, to achieve the effect of accelerating the breeding process, good repeatability, and stable and reliable test results

Active Publication Date: 2015-04-29
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of production, restorer genes are the basis for the utilization of heterosis in peppers, but the biggest obstacle to the application of CMS male sterile lines in peppers is that the fertility restorer genes are not widely distributed. Zou School, 1991; Liu Jinbing, et al., 1999), most bell peppers do not contain restorer genes
The three-line breeding cycle is long, and if molecular markers closely linked to restorer genes or sterility genes can be found, it will help shorten the breeding process

Method used

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  • Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker
  • Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker
  • Method for detecting SSR molecular marker of pepper male sterility restoring gene as well as kit of SSR molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, (capsicum male sterility restoration gene Rf development of co-segregated molecular markers)

[0040] Pepper materials described in this example: pepper sterile line 131BC5A; restorer line 139D-21-3; 131BC5A x 139D-21-3 F 1 Generation single plant; 131BC5A x 139D-21-3 F 2 separate groups;

[0041] Parent material and hybridization method:

[0042] ①Parent material:

[0043] Restorer line 139D-21-3: In the spring of 2009, an excellent single plant was discovered from the farm field of Yangdu Village, Xucun Town, Haining City, Zhejiang Province. After obtaining consent, the fruit was harvested and the seeds were collected, and the seeds were purified by self-fertilization for multiple generations. . Restoration line 139D-21-3 can be purchased by the public from Zhejiang Academy of Agricultural Sciences before the date of this patent application. Zhejiang Academy of Agricultural Sciences has distributed this biological material to the public since Octobe...

Embodiment 2

[0054] Embodiment 2, (the capsicum male sterility restoration gene is detected with the marker primer of the present invention)

[0055] Pepper materials described in this example: Pepper sterile line control material: 131BC5A, numbered 1; restorer control material: 139D-21-3, numbered 2; optional pepper recovery materials: numbered 5, 6 and 7; optional pepper Sterile material: numbered 3, 4, 8 and 9;

[0056] The detection method is carried out according to the following steps:

[0057] 1. Extraction of genomic DNA of pepper: Take 0.02-0.05 grams of the young leaves of the pepper samples to be tested at the seedling stage, extract the DNA according to the conventional CTAB method, and store them separately for later use;

[0058] 2. PCR amplification of genomic DNA: Add 1 μL of the genomic DNA of each pepper sample extracted in step (1) to each PCR tube, and then add 5 μL of 2× Taq PCR MasterMix in sequence, and 0.25 μL of the upstream and downstream primers of pep43 or pep2...

Embodiment 3

[0061] Embodiment 3, (use kit of the present invention to the detection of gene restoration of different capsicum materials)

[0062] Pepper materials described in this example: Pepper male sterile line control material: 131BC5A, numbered 1; 11. Optional pepper sterile material: number 4, 6 and 7; maintainer line N(msms) 08-131: number 10; local pepper material 08032: number 12;

[0063] The detection method is carried out according to the following steps:

[0064] 1. Extraction of genomic DNA of pepper: Take 0.02-0.05 grams of the young leaves of the pepper samples to be tested at the seedling stage, extract the DNA according to the conventional CTAB method, and store them separately for later use;

[0065] 2. PCR amplification of genomic DNA: use the primers and reagents provided in the kit for PCR amplification, the amplification volume is 10 μL; add 1 μL of DNA template, 5 μL of 2× Taq PCR MasterMix, 0.25 μL of upstream and downstream primers, Add sterile pure water to 10 ...

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Abstract

The invention relates to the field of assistant breading in biotechnologies, in particular to an SSR marker linked with a pepper male sterility restoring gene Rf as well as an application of the SSR marker to selection of pepper male sterility breeding materials. The molecular marker closely linked with the pepper male sterility restoring gene is a pep43 marker or a pep20 marker. The development of the SSR molecular marker can accelerate the breeding process of the pepper male sterility, improve the breeding effect and provide a theoretical basis for classification and breeding of three systems of pepper CMS (cytoplasmic male sterility); an independently developed SSR primer and a conventional laboratory PCR (polymerase chain reaction) technology are adopted, and plants with restoring genes are selected through detection of pepper plants. The method is used for detecting genetic materials, is convenient and good in repeatability and can perform detection all the year round. According to a kit, the primer and related reagents for detection are integrally assembled in the kit, operations are convenient to implement, and detection results are stable and reliable.

Description

technical field [0001] The invention relates to the field of biotechnology-assisted breeding, in particular to a pepper male sterility restoration gene Rf Linked SSR markers and their application in selection of pepper male sterile breeding materials. Background technique [0002] chili( Capsicum annuum L.) is a worldwide vegetable and plays an important role in vegetable production. Male sterility is one of the important traits in crop breeding. Capsicum is a cross-pollination crop with very obvious heterosis. The use of male sterility traits can solve the problem of artificial detasseling of heterosis and reduce production costs. Pepper male sterility can be divided into Cytoplasmic male sterility (CMS) and Genetic male sterility (GMS), among which CMS requires simultaneous breeding of "three lines", including fertile cytoplasm The maintainer line N (msms); the sterile cytoplasmic male sterile line S (msms); the fertility control restorer line S (MsMs) or N (MsMs). ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 叶青静杨悦检阮美颖王荣青周国治姚祝平李志邈万红建程远
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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