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EST-SSR marker development and application of rhododendron pulchrum sweet

A rhododendron and splendid technology, applied in the field of SSR molecular marker development and cross-species amplification application, can solve the problems of limitation and limited microsatellite markers, and achieve the effect of high versatility

Active Publication Date: 2019-03-19
HUANGGANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the published microsatellite markers available for genetic research in Rhododendron splendens are very limited, which limits the application of molecular marker-assisted breeding of new varieties of Rhododendron splendens
At present, there is no report on the development of SSR using Rhododendron splendens transcriptome data

Method used

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  • EST-SSR marker development and application of rhododendron pulchrum sweet
  • EST-SSR marker development and application of rhododendron pulchrum sweet
  • EST-SSR marker development and application of rhododendron pulchrum sweet

Examples

Experimental program
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Effect test

Embodiment 1

[0040] [Example 1] Development of EST-SSR primers for Rhododendron splendens with high polymorphism

[0041] Based on the high-throughput sequencing of transcriptome provided by the present invention, combined with bioinformatics methods for SSR sequence search and SSR marker primer design and verification, the specific implementation methods are as follows:

[0042](1) Screening of Unigene sequences rich in SSR sites: Total RNA was extracted from Rhododendron splendens, a transcriptome sequencing library was constructed, and high-throughput sequencing was performed using Illumina next-generation sequencer. The sequencing data was assembled after strict filtering to obtain Unigene, which was used as background data for subsequent SSR primer development. Use MISA software to search for SSR sites in Unigene. The search criteria are: dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides. The minimum repeat times are 10, 8, 6, respectively. 5, 4 t...

Embodiment 2

[0048] [Example 2] Application of 15 pairs of EST-SSR primers in the study of genetic diversity of Rhododendron splendens

[0049] (1) DNA extraction: A total of 35 individuals of Rhododendron splendens were collected in the Dabie Mountains of Hubei Province (the collection location is distributed at 114.56°-115.043°E east longitude, 31.03°-31.68°N north latitude, and 220-650m above sea level), and the numbers are numbered For JXDJ1-JXDJ30. The genomic DNA of the leaf tissue was extracted by 3×CTAB method, and the quality was detected by 1% agarose gel electrophoresis and the concentration was determined by ultraviolet spectrophotometry. After dilution to 100ng / μl, it was stored in a -20°C refrigerator for later use.

[0050] (2) PCR amplification: 20μl reaction system contains: ddH 2 O 15.7μl, 10×Buffer (containing Mg 2+ ) 1.5 μl, dNTPs (10 mM) 0.2 μl, 10 μM forward and reverse primers 0.2 μl each, Taq DNA polymerase 0.2 μl, DNA template (50ng / μl) 1 μl. The reaction progra...

Embodiment 3

[0056] [Example 3] Detection of 15 pairs of EST-SSR primers for cross-species amplification in 3 related species of Rhododendron splendens

[0057] The general detection steps of EST-SSR primers in three Rhododendron splendens relatives:

[0058] (1) Genomic DNA extraction: 3 × CTAB method was used to extract genomic DNA from fresh leaf tissue of three Rhododendron splendens relatives: liquid nitrogen grinding, phenol / chloroform (volume ratio 1:1) extraction, isopropanol precipitation , 70% ethanol washing, and RNase treatment, the extracted DNA was dissolved in 50 μL TE solution.

[0059] (2) PCR amplification. Use a 15 μl reaction system containing ddH 2 O 11.6μl, 10×Buffer (containing Mg 2+ ) 1.5 μl, dNTPs (10 mM) 0.2 μl, forward and reverse primers (10 μM) 0.2 μl each, Taq DNA polymerase (2.5U / μl) 0.3 μl, DNA template (50ng / μl) 1 μl. The reaction program was set as follows: pre-denaturation at 94°C for 5 min, 35 amplification cycles (denaturation at 94°C for 40 sec, am...

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Abstract

The invention discloses EST-SSR marker development and application of rhododendron pulchrum sweet and belongs to the biotechnical field. The primer is obtained by developing a rhododendron pulchrum sweet transcriptome. Based on RNA-seq, a lot of transcriptome sequencing data is screened to obtain a sequence rich in SSR loci and the primer marked by SSR is designed, so that the obstacles that SSR molecular markers of existing hododendron pulchrum sweet are few and the developing efficiency is low are overcome. The universality and effectivenss of the primer are detected by three sibling speciesprimers, thereby laying a foundation for analytical investigation of genetic diversity, researches on genetic linkage map structure, flower-like key gene positioning, molecular marker assistant breeding of hododendron pulchrum sweet and other ericaceae plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the development of SSR molecular markers and the application of cross-species amplification based on Rhododendron splendens transcriptome data. Background technique [0002] Rhododendron pulchrum Sweet is an evergreen shrub belonging to the Rhododendron family (Ericaceae). The flowering period is mainly from February to April. It has bright colors, strong adaptability and rapid growth. In the landscaping and the beauty of the greening in front of and behind the house. The dried leaves of Rhododendron splendens can eliminate phlegm, relieve cough, etc., and have important medicinal value. Splendid Rhododendron contains a large amount of plant volatile organic compounds such as natural pigments, flavonoids, terpenes, aromatics and aldehydes. For a long time, breeding scholars have done a lot of work on the regulation of the flowering period of Rhododendron splendens, prov...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王书珍李志良金卫斌张明菊郭旭栋胡雨晴夏奥蕾
Owner HUANGGANG NORMAL UNIV
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