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EST-SSR molecular marker developed based on lycoris aurea transcriptome sequence and application thereof

A technology of transcriptome sequence and labeling, which can be used in recombinant DNA technology, DNA/RNA fragments, determination/inspection of microorganisms, etc., and can solve problems such as non-specific molecular markers.

Inactive Publication Date: 2018-08-03
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no specific molecular marker for Hudixiao. Therefore, the development of Hudixiao EST-SSR markers has become an urgent problem to be solved in the research of Hudixiao germplasm resources and the improvement of Hudixiao varieties in the future.

Method used

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  • EST-SSR molecular marker developed based on lycoris aurea transcriptome sequence and application thereof
  • EST-SSR molecular marker developed based on lycoris aurea transcriptome sequence and application thereof
  • EST-SSR molecular marker developed based on lycoris aurea transcriptome sequence and application thereof

Examples

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example 1

[0021] Example 1 : 20 SSR markers developed from the Smiley Transcriptome were obtained by the following method:

[0022] (1) Obtain the EST sequence according to the transcriptome sequencing results of Hudi Xiao, use the software Microsatellite (MISA) to find the SSR sequence and design primers. The principle of primer design is that the length of the PCR product is 100-350bp; the GC content is 40-70%, and the maximum Suitable for 50%; annealing temperature 50-65 ℃; primer length: 19-22bp, the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.;

[0023] (2) Harvest the young leaves of Hudixiao, and the information on the germplasm resources of Hudixiao is shown in Table 2. The DNA of the sample to be tested was extracted by the Biotech Plant Genomic DNA Rapid Extraction Kit, and the operation was carried out with reference to the instructions of the kit. Dilute it by 50 times, check the DNA concentration by spectrophotometer, check the quality by electrophor...

example 2

[0037] Example 2: Genetic diversity analysis of 20 EST-SSR markers applied to the material in Table 2

[0038] (1) Extract the genomic DNA of the material in Table 2, and adjust the DNA concentration to 10ng / μL;

[0039] (2) Using the above DNA as a template, use the primers shown in SEQ ID NO. 1 to 40 in Sequence Table 1 to carry out PCR amplification, and add 2.0 μL of 10x Taq Buffer (containing Mg to the 20 μL reaction system respectively). 2+ ), 1.6μ of 2.5nM dNTP, 5units / μL Taq DNA polymerase 0.1μL, forward primer 4pmol, reverse primer 4pmol, 10ng / μL DNA template 1μL, ddH 2 O supplement the system to 20 μL. DNA amplification was performed using an Eppendorf Mastercycler. The specific reaction procedure was: pre-denaturation at 94°C for 3 min, followed by 35 cycles of 94°C (30S) / 58°C (30s) / 72°C (30S), and a final extension at 72°C for 10 min;

[0040] (3) The PCR amplification product in step (2) was detected by 8% polyacrylamide gel electrophoresis, the voltage was 180V...

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Abstract

The invention discloses a set of EST-SSR molecular marker developed based on a lycoris aurea transcriptome sequence. The EST-SSR molecular marker comprises 20 pairs of primers, wherein nucleotide sequences are as shown in a sequence table SEQ ID NO. 1-40. The 20 pairs of EST-SSR marked primers are designed according to the transcriptome sequence, are proved to be definite SSR markers through genome DNA amplification and sequencing, have advantages of rich polymorphism, stable amplification, good repeatability, codominance and convenience in detection and the like, and can better distinguish the diversity of genetic germplasm of lycoris aurea. The set of markers fills up the lack of specific codominant markers of lycoris aurea, and can be applied to fields such as genetic diversity of germplasm resources, varietal purity identification, variety genetic lineage analysis and assisted breeding of lycoris aurea, thereby greatly promoting research, application and development of germplasm resources of lycoris aurea.

Description

technical field [0001] The invention relates to molecular marker technology, in particular to 20 EST-SSR molecular markers and applications thereof. Background technique [0002] It is a perennial herb of Amaryllidaceae (Amaryllidaceae), which has important medicinal value and ornamental value. It not only contains lycorine, galanthamine, narciclasine and other amaryllidaceae alkaloids with specific pharmacological activity, but also has important ornamental value. Observable leaves. However, there are great differences in the germplasm resources of different sources, not only in the external morphology and karyotype, but also in the alkaloid content. The use of molecular markers to carry out the fingerprint analysis of Kudixiao germplasm resources can not only investigate the diversity of Kudixiao germplasm resources, but also has important significance for the efficient utilization and development of Kudixiao germplasm resources in the future, such as the high alkaloid c...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 汪仁江玉梅徐晟王蓉贺佳王松凤周佳宇
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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