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Wintersweet est-ssr marker and its application in genetic diversity analysis

A technology of genetic diversity and wintersweet, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of limiting the analysis of genetic diversity and species identification and protection of wintersweet

Active Publication Date: 2020-06-16
ZHEJIANG FORESTRY UNIVERSITY
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  • Abstract
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Problems solved by technology

[0004] At present, there are relatively few reports on the development of SSR primers and related genetic studies of Wintersweet, which greatly limits the genetic diversity analysis and variety identification and protection of Wintersweet.

Method used

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  • Wintersweet est-ssr marker and its application in genetic diversity analysis
  • Wintersweet est-ssr marker and its application in genetic diversity analysis
  • Wintersweet est-ssr marker and its application in genetic diversity analysis

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Embodiment Construction

[0042] (1) Transcriptome sequencing

[0043] Collect about 0.5 g of fresh and tender leaves of wild wintersweet wintersweet, wrap them in tin foil, place them in liquid nitrogen for quick freezing, and store them at -80°C for later use. Total RNA of Wintersweet was extracted using RNAiso Plus (TaKaRa). Nanodrop and Agilent 2100 were used to detect the concentration and quality of total RNA before sequencing. OD260 / 280 and OD260 / 230 were between 1.8-2.2, 28S:18S>1.3, RIN>7, no degradation and no pollution. Use QUBIT RNA ASSAY KIT to accurately quantify the starting total RNA, and the amount of total RNA is 0.1-1 μg. Use Beads with Oligod(T) to enrich and purify mRNA in total RNA, add fragmentation buffer to fragment mRNA into short fragments, use mRNA as a template, and use random hexamers to synthesize the first cDNA strand ; Then add buffer, dNTPs, RNase H and DNA polymerase I to synthesize the second cDNA strand; after the kit is purified and eluted with EB buffer, do end ...

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Abstract

The invention provides a development method of an EST-SSR molecular marker of an endangered plant sinocalycanthus chinensis. The development method mainly comprises the following steps: identifying and screening an EST sequence and an SSR site of the sinocalycanthus chinensis, thus obtaining an EST-SSR molecular marker primer sequence. According to the development method provided by the invention, 200 sequences containing different repetitive units are searched from data of a sinocalycanthus chinensis transcriptome, and software analysis and molecular marker screening are combined to obtain 23 EST-SSR markers with abundant polymorphism. The EST-SSR marker can be applied to genetic diversity analysis, germplasm identification, variation and evolution researches, molecular marker assisted breeding and the like of different species and different species groups of the endangered plant sinocalycanthus chinensis. The EST-SSR marker has good repeatability and high reliability and is a very effective EST-SSR molecular marker.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a molecular marker technology based on transcriptome high-throughput sequencing for the entire genus of Wintersweet. Background technique [0002] Sinocalycanthus chinensis (Cheng et S.Y.Chang) belongs to the genus Wintersweet, which is a relict plant of the Tertiary period. It is mainly distributed in Zhejiang Province, China (Lin'an City and Tiantai County), and belongs to the second-class national key protection. Species have great scientific research value in the study of flora. Wintersweet is a dicotyledonous plant, a shrub, with clustered stems, terminal flowers, and no scales in the buds. It has great ornamental value and medicinal value. There are very few wild resources of wintersweet. Because its living environment is affected by natural disasters and human disturbance, it is divided into many small populations, so that there are different distances between different popul...

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 赵宏波董彬张超付建新周莉花黄耀辉吴静
Owner ZHEJIANG FORESTRY UNIVERSITY
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