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Method for identifying male sterile cytoplasmic type of maize based on KASP technology

A technology of corn and technology, applied in the field of crop molecular biology, can solve the problems of limited range of identification materials, inability to achieve high-throughput, automatic identification, and long identification period

Active Publication Date: 2018-09-04
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The identification period of the first three methods is long, and the scope of identification materials is limited; there is currently no reported identification method for the development of InDel sites in the chloroplast genome based on the PCR method, and they are all based on electrophoresis platforms such as agarose and polyacrylamide. Throughput, automated identification

Method used

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  • Method for identifying male sterile cytoplasmic type of maize based on KASP technology
  • Method for identifying male sterile cytoplasmic type of maize based on KASP technology
  • Method for identifying male sterile cytoplasmic type of maize based on KASP technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Acquisition of Specific Primers Suitable for Distinguishing Maize Male Sterile Cytoplasmic Types

[0044] 1. Sample selection: 170 widely representative maize inbred lines were selected for whole genome sequencing. These 170 samples include common corn, waxy corn, sweet corn, popcorn and other corn types; including all heterosis groups in my country, Tang Sipingtou, Lvda Honggu, Reid, Lanka, improved Reid, improved Lanka, P group, landraces, and T, C, S three types of cytoplasmic sterile materials.

[0045] 2. Sample preparation: The seeds of 170 corn samples were germinated in the incubator for 5 days. The first 3 days were under no light conditions, and the next 2 days were under light conditions (that is, sufficient light was given after emergence). For each sample, 30 green seedling leaves were selected and mixed, and fully ground under liquid nitrogen conditions. Total DNA was extracted by CTAB method, and RNA was removed. Detect the quality of the extr...

Embodiment 2

[0053] Example 2 High-throughput identification method of maize male sterile cytoplasmic type based on KASP technology

[0054] Purpose of the experiment: To identify the male sterile cytoplasmic types of 96 maize inbred germplasm materials.

[0055] 1. DNA extraction of samples of corn inbred lines to be identified: 50 seeds were randomly selected for each sample, seedlings were germinated, and sufficient light was given to form green seedlings. The method of DNA extraction was mixed plant DNA extraction. For each sample, 30 green leaves of individual plants were randomly selected from 50 individual plants and mixed. The specific steps of DNA extraction were carried out in accordance with the molecular identification standard of corn DNA (Wang Fengge et al., 2014, Maize Variety Identification Technical Regulations SSR Marking Method, Agricultural Industry Standard of the People's Republic of China). Dilute the DNA to form a working solution with a concentration of 20ng / μL. ...

Embodiment 3

[0062] Example 3 Using the specific primers provided by the present invention to identify whether the maize hybrid material is male sterile seed production and which type of sterile cytoplasm it belongs to

[0063] The purpose of the experiment: To identify whether the 96 hybrids are hybrids produced by cytoplasmic sterile seed production, and which type of sterile cytoplasm they belong to, the specific method is as follows:

[0064] 1. DNA extraction of samples of corn hybrids to be identified: randomly select 50 seeds from each packaging bag, germinate seedlings, and give sufficient light to form green seedlings. The method of extracting DNA from mixed plants was adopted for DNA extraction, and the green leaves of 30 individual plants were randomly selected from 50 individual plants for each sample, forming 96 samples to be tested. The specific steps of DNA extraction were carried out in accordance with the standards for molecular identification of corn DNA (Wang Fengge et a...

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Abstract

The invention provides a method for identifying a male sterile cytoplasmic type of maize based on a KASP technology. Chloroplast genome maize male sterile S-type cytoplasmic identification specific primers comprise 19 pairs, including 17 SNPs and 2 InDel site primers. C-type cytoplasmic identification specific primers comprise 10 pairs, including 9 SNPs and 1 InDel site primers. T-type cytoplasmicidentification specific primers comprise 7 pairs of SNP primers. The method can be used for identifying germplasm materials of male sterile cytoplasm and normal cytoplasm of corn, identifying hybridsof sterile seeds and conventional seeds, and identifying the purity of sterile materials. The method provides the technical support for corn cytoplasmic sterile seed production and breeding of sterile seeds, provides a strong guarantee for seed production of maize three lines and expands the range of maize cytoplasm hereditary characters through the range of marker sites of corn in the genome level.

Description

technical field [0001] The invention belongs to the technical field of crop molecular biology, and in particular relates to a method for identifying maize male sterile cytoplasmic types based on KASP technology. Background technique [0002] Maize is the first crop to realize the use of heterosis, and the breeding and utilization of its hybrids is known as a revolution in crop production. There are two methods of corn hybrid seed production: conventional and male sterile. The use of male sterility to prepare corn hybrids not only saves a lot of labor and reduces costs, but also improves the purity of hybrids and increases yield. [0003] Maize male sterility (male sterility, MS) refers to the phenomenon that the abnormal development of stamens in maize produces non-functional pollen, while the normal development of pistils can normally pollinate and set fruit. Male sterility in maize can be roughly divided into two types: cytoplasmic male sterility (CMS) and genetic male st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113
Inventor 王凤格田红丽王蕊赵久然杨扬许理文易红梅葛建镕刘亚维
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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