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Lycoris spp. plant fluorescence EST-SSR molecular marker and application thereof

A technology of Lycoris plants and molecular markers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of limiting Lycoris resources identification and molecular genetics research, and achieve automation High degree of accurate identification of the effect of the analysis

Active Publication Date: 2020-05-08
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Resource identification and molecular genetics studies of Lycoris are limited

Method used

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  • Lycoris spp. plant fluorescence EST-SSR molecular marker and application thereof
  • Lycoris spp. plant fluorescence EST-SSR molecular marker and application thereof
  • Lycoris spp. plant fluorescence EST-SSR molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Design of EST-SSR Marker Primers for Lycoris

[0060] The EST-SSR sequences of Lycoris genus used in the development of primers come from 404,481 Unigenes that were sequenced and sequenced with Huanjinhua, Lycoris chinensis, Lycoris chinensis, Hudixiao, Lycoris longifolia and Lycoris syringum in the early stage, and were analyzed by perl script MISA software The SSR loci in Unigene were analyzed. After the primer design was completed, it was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and a total of 30 pairs of EST-SSR primers were synthesized. Among them, some primers (Table 2 provides the specific primer sequences involved in the examples and comparative examples. Including QZ155, QZ171, QZ173, QZ175, QZ177, QZ203, QZ207, QZ209, QZ211) sequences are shown in Table 2:

[0061] Table 2 Lycoris EST-SSR primers

[0062]

Embodiment 2

[0064] Screening of Lycoris EST-SSR Markers, Analysis of Genetic Diversity and Detection of Capillary Electrophoresis

[0065] Screening of Lycoris EST-SSR Markers and Results of Capillary Electrophoresis

[0066] Using the SSR site information obtained by MISA, combined with Primer3.0 to design SSR primers in batches, 30 pairs were selected for primer effectiveness and polymorphism screening. For primers with good amplification effect and clear bands, synthesize fluorescently labeled EST-SSR primers, and perform polymorphism analysis among different species.

[0067] Using 4 pairs of Lycoris EST-SSR primers, samples from 16 species were analyzed, and the optimized annealing temperature and amplified fragment size are shown in Table 3. Synthesize fluorescently labeled primers, and use fluorescently labeled primers for PCR. The reaction system is 25 μL, including: 1 μL genomic DNA, 3.2 pmol (0.5 μL each) forward and reverse primers, 10 mM (0.5 μL) dNTP, 2.5 μL 10× PCRBuffer, ...

Embodiment 3

[0078] Establishment of Fingerprint Library of 16 Lycoris Species by Capillary Electrophoresis

[0079] The screened 4 pairs of fluorescent EST-SSR primers were used to amplify 16 Lycoris species, and the fragment sizes of alleles of different materials at different sites were accurately obtained (Table 5). For example: Lycoris amplified fragments at SSR site QZ209 are 206, 212 and 218bp in size. The amplification band patterns of each pair of primers ranged from 4 to 13, with an average of 8.2. According to the peak diagram of the amplification results of the 4 pairs of core primers, the size of the band can be read accurately, and all species can be distinguished. 4 pairs of primer combinations (QZ209, QZ155, QQZ175, QZ171) can completely distinguish 16 species (Table 5). Among them, the number of polymorphic sites of primer QZ209 reaches 12, which can be distinguished: Lycoris longum, Lycoris yellow long tube, Chinese Lycoris, Lycoris rose, Lycoris rose, Red and blue Lyco...

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Abstract

The invention relates to a lycoris spp. plant fluorescence EST-SSR molecular marker and application thereof, and belongs to the technical field of molecular markers. The molecular marker comprises QZ209, QZ155, QZ175 and QZ171. The molecular markers QZ209, QZ155, QZ175 and QZ171 can completely distinguish 16 pieces of lycoris spp. species, wherein a number of polymorphism sites of a primer of theQZ209 is 12, a PIC (polymorphism information content) value is 0.92, and different species of materials of 12 pieces of lycoris spp. can be distinguished through independent application of the QZ209,the QZ209 can be used as an excellent SSR molecular marker to be applied to subsequent classification and identification work of the lycoris spp. species, and a scientific basis is provided for the protection and development of lycoris spp. resources.

Description

technical field [0001] The invention relates to the technical field of molecular markers, in particular to fluorescent EST-SSR molecular markers of Lycoris plants and applications thereof. Background technique [0002] Lycoris spp. is an important medicinal and ornamental plant native to my country. Its flower color and flower type are changeable and highly ornamental. The bulbs contain lycoris, galantamine and other alkaloids. , has the important functions of treating myasthenia gravis, senile dementia (Alzheimer's disease, AD), anti-tumor and anti-cancer, etc., and has extremely high economic value. The flowers and leaves of Amaryllis plants have high ornamental value, are prone to variation, and have high interspecific hybridization affinity. It takes 5 years to form a flowering bulb. Only by the appearance shape, color and size of leaves, bulbs and flowers, there will be errors in variety identification and classification, and confusion is likely to occur in sales and pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/13C12Q2600/156C12Q2565/125C12Q2563/107
Inventor 李青竹蔡友铭张永春杨柳燕许俊旭孙翊李心周晓慧王桢
Owner SHANGHAI ACAD OF AGRI SCI
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