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Molecular marker primer for detecting aneuploid hybrid progeny plants, and detection method of aneuploid hybrid progeny plants

A technology of molecular markers and aneuploidy, applied in the field of plant genetics and breeding in biotechnology, can solve the problems of restricting the wide application of emerging molecular karyotype analysis technology and high detection cost

Active Publication Date: 2015-04-22
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high cost of detection limits the widespread application of the above-mentioned emerging molecular karyotyping techniques

Method used

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  • Molecular marker primer for detecting aneuploid hybrid progeny plants, and detection method of aneuploid hybrid progeny plants
  • Molecular marker primer for detecting aneuploid hybrid progeny plants, and detection method of aneuploid hybrid progeny plants
  • Molecular marker primer for detecting aneuploid hybrid progeny plants, and detection method of aneuploid hybrid progeny plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Preliminary detection of ploidy level of hybrid progeny plants

[0036] Take 50 mg of young leaves of progeny plants to be tested, put them on ice, add 1 mL of refrigerated nucleus isolation buffer, wherein, the nucleus isolation buffer is 45mM MgCl2 6H2O, 20mM MOPS, 30mM sodium citrate, 0.5% Triton X-100 , 1% PVP-10, pH 7.0; then quickly chopped into pieces with a single-sided blade for 60 seconds, the separation buffer containing nuclei was filtered through a gauze with a pore size of 40 μm, and a concentration of 50 μg / mL PI (Propidium Iodide, iodine) was added to the filtrate Propidium staining solution) solution, mixed evenly, wherein the PI solution contains RNase, the concentration of RNase in the PI solution is 50 μg / mL, then incubated in an ice bath for staining for 30min, and then used a flow cytometer (Partec, Germany) , model: CyFlow PA flow cytometer) for nuclear DNA content analysis.

[0037] The nuclei of both parents (ie, father and mother) were used...

Embodiment 2

[0084] 1. Preliminary detection of ploidy level of hybrid progeny plants

[0085] Same as Example 1.

[0086] 2. Extract the DNA of the father, mother and hybrid offspring

[0087] Same as Example 1.

[0088] 3. Primer screening

[0089] Based on the set marker requirements, through screening, the physical location of the marker site is close to the centromere, the parental differences and co-dominant segregation molecular marker primers, the primer PMGC-2607 located on chromosome VIII of poplar (see Table 3) is an example analysis, which is derived from the database of the International Poplar Genome Consortium (http: / / web.ornl.gov / sci / ipgc / ssr_resource.htm).

[0090] 4. PCR amplification and capillary fluorescence electrophoresis analysis

[0091] 4-1. Use PMGC-2607 primers to perform PCR amplification on the DNA of the hybrid parent and perform capillary fluorescence electrophoresis on the amplified product, wherein, the PCR reaction procedure and capillary fluorescence...

Embodiment 3

[0109] 1. Preliminary detection of ploidy level of hybrid progeny plants

[0110] Same as Example 1.

[0111] 2. Extract the DNA of the father, mother and hybrid offspring

[0112] Same as Example 1.

[0113] 3. Primer screening

[0114] Based on the set marker requirements, through screening, the physical location of the marker site is close to the centromere, the parental differences and co-dominant segregation molecular marker primers, primer LG-10-1788 located on chromosome X of poplar (See Table 3) for example analysis, derived from the report of Yin et al. (2009).

[0115] Table 3 The SSR primers suitable for the identification of chromosome number VIII and X in the offspring of ‘Pulus maoxin’בYinzhong’

[0116]

[0117] 4. PCR amplification and capillary fluorescence electrophoresis analysis

[0118] 4-1. Use LG-10-1788 primers to perform PCR amplification on the DNA of the hybrid parent and perform capillary fluorescence electrophoresis on the amplified produc...

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Abstract

The invention discloses a detection method of aneuploid hybrid progeny plants. The method comprises the following steps: firstly performing preliminary assessment of ploidy levels of hybrid progeny plants; by using progeny plants and parental DNAs as a template, performing PCR amplification respectively; respectively performing fluorescence capillary electrophoresis on amplified products; finally, by taking parental genes as reference and combining the preliminarily assessed ploidy levels of the plants, identifying the number and height of obtained allele peaks, comparing the composition conditions of the alleles of the progeny plants, and if the number of marker sites positioned on a certain chromosome is more than or less than the expected number, determining to be aneuploid plants. The method provided by the invention can be used for high-throughput, rapid and accurate detection of aneuploid plants, and has an important significance on the study on aneuploidy hybrid genetic improvement and chromosomal operation. Compared with the traditional karyotype analysis method, the method has the advantages of low cost, easiness in operation, rapid detection speed, strong reliability and the like, and is suitable for analysis of large-scale mass materials.

Description

technical field [0001] The invention relates to a method for detecting chromosome composition of aneuploid hybrid progeny plants, belonging to the field of plant genetics and breeding in biotechnology. Background technique [0002] The utilization of chromosome number variation (polyploidy and aneuploidy) is an important approach for plant genetic improvement, and different chromosome number and chromosome composition can produce individual materials with significant phenotypic trait variation. Classic cases of chromosome aneuploidy variation in higher plants such as Nicotiana monosomy series and Datura stramonium trisomy series all show obvious variation in plant shape or fruit shape. Therefore, aneuploid breeding is an important area of ​​plant genetic improvement. [0003] To analyze the chromosome composition of polyploids and aneuploids, traditional chromosome banding analysis, fluorescence in situ hybridization (Fluorescent in situ Hybridization, FISH) and other karyo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 王君尚峰男田菊
Owner BEIJING FORESTRY UNIVERSITY
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