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Cypress microsatellite molecular marker combination, primer screening method and its application

A molecular marker and screening method technology, applied in biochemical equipment and methods, microbial assay/inspection, recombinant DNA technology, etc., can solve the problem of lack of effective means of cypress germplasm resources

Inactive Publication Date: 2021-01-08
BEIJING FORESTRY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In order to solve the problem of the lack of effective means for identifying cypress germplasm resources in the prior art, the purpose of the invention is to provide a group of high-efficiency, operational methods that can be directly applied to intraspecific and interspecific cypress, and have good versatility and practicability. Simple, high detection accuracy, stable and efficient chloroplast polymorphic microsatellite molecular marker, its screening method and amplification primers, variety identification method and parent-origin relationship identification method

Method used

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  • Cypress microsatellite molecular marker combination, primer screening method and its application
  • Cypress microsatellite molecular marker combination, primer screening method and its application
  • Cypress microsatellite molecular marker combination, primer screening method and its application

Examples

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Effect test

Embodiment 1

[0246] Embodiment one kind of polymorphism primer screening method of cypress chloroplast microsatellite molecular marker

[0247] 1. Screening of chloroplast microsatellite molecular markers

[0248] From the GenBank public database (https: / / www.ncbi.nlm.nih.gov / genbank / ), download the full sequence of the chloroplast genomes of the following six species of cypress: giant cypress (Cupressus gigantean), Mediterranean cypress (Cupressus sempervirens), North American cherry round Cypress (Juniperus monosperma), Bermuda juniper (Juniperus bermudiana), Rocky Mountain juniper (Juniperus scopulorum) and North American juniper (Juniperus virginiana), using GMATA2.1 software to analyze the full sequence of the chloroplast genome of the above six species of cypress, and screen microsatellite molecular markers, namely SSR Loci, the screening conditions are that the number of repeats is greater than 5, the length of the repeat fragment is between 2bp and 10bp, and the total length is les...

Embodiment 2

[0351] Embodiment 2. A method for analyzing cypress genetic diversity using the polymorphic primers of the cypress microsatellite molecular markers provided in Example 1.

[0352] Utilize the polymorphic primers of N2, N8, N11, N13, N20, the polymorphic primers of the chloroplast microsatellite molecular markers of N27 screened out by the embodiment one, adopt the chloroplast microsatellite molecular markers of 192 arborvitae leaf samples to be amplified by the three-primer method , the samples came from the oriental cypress seed orchard in Jia County, Henan Province, the molecular size of the fluorescent PCR product was detected by capillary electrophoresis, and the original data were collected by GeneMarker V2.4.0 software and the genetic diversity analysis of the tested samples was carried out. The specific operation is as follows:

[0353] 1. Extraction of Chloroplast DNA from a Single Sample

[0354] The improved CTAB method was used to extract the chloroplast DNA of 192...

Embodiment 3

[0391] Example 3 A method for identifying the genetic relationship of cypress using the polymorphic primers of the cypress microsatellite molecular marker provided in Example 1.

[0392] Utilize the polymorphic primers of N2, N8, N11, N13, N20, the polymorphic primers of the chloroplast microsatellite molecular markers of N27 screened out by the embodiment one, adopt the chloroplast microsatellite molecular markers of 192 arborvitae leaf samples to be amplified by the three-primer method , the samples came from the oriental cypress seed orchard in Jiaxian County, Henan Province, the molecular size of the fluorescent PCR product was detected by capillary electrophoresis, and the original data were collected by GeneMarker V2.4.0 software and the genetic diversity analysis of the tested samples was carried out. The specific operation is as follows:

[0393] 1. Extraction of Chloroplast DNA from a Single Sample

[0394] The improved CTAB method was used to extract the chloroplast...

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Abstract

The invention discloses a cypress microsatellite molecular marker combination, and a primer combination and screening method and application thereof, and belongs to the technical field of forest molecular biology. In the application, a method for identifying the polymorphism of cypress chloroplast microsatellite molecular marker primers comprises the steps that an M13 tail sequence is added at the 5' terminal of a forward primer of a designed primer pair, then the designed primer pair is mixed with a specific reverse primer, a general M13 primer with a fluorescence marker and a genome DNA, PCR amplification and electrophoretic separation sequencing are carried out, and polymorphic primers of cypress microsatellite molecular markers are obtained. A method for identifying the genetic relationship of cypress comprises the steps that the genome DNA of cypress is extracted, PCR amplification and electrophoretic separation are carried out with the polymorphic primers of the cypress microsatellite molecular markers, capillary electrophoresis sequencing is carried out, STR polymorphism is counted, the polymorphic primers can reflect the genetic information of cypress plasmon and can be used for researches such as genetic resource classification, genetic diversity analysis and the like.

Description

technical field [0001] The invention relates to the field of forestry molecular biology technology, in particular to cypress molecular marker technology, more specifically to a cypress microsatellite molecular marker and its screening method, and its application in the fields of identification of cypress parentage relationship, variety identification and assisted breeding. Background technique [0002] Many species of Cupressaceae are the main wood raw materials, and are widely used in gardening, medicine and other fields. The whole body of cypress is a treasure, and its resin, tree oil, fruit, branches and leaves can all be used as medicine. The seeds and leaves of orientalis arborvitae are the most widely used clinically. Seed kernel: sweet, flat, can nourish the heart and calm the nerves, stop sweating, and moisten the intestines. Branches and leaves: bitter, slightly cold, cooling blood to stop bleeding, hair growth and black hair, can cure hematemesis, dysentery, hemo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 黄丽莎张玉平曹均毛建丰刘国彬
Owner BEIJING FORESTRY UNIVERSITY
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