Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof

A technology of molecular markers and specific primers, used in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc.

Inactive Publication Date: 2010-03-31
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] As of January 16, 2009, GenBank (http: / / www.ncbi.nlm.nih.gov / dbEST / ) released a total of 11,748 Salvia ESTs, including 10,288 Salvia ESTs, but currently There have been no reports of developing SSR markers using these ESTs

Method used

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  • Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof
  • Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof
  • Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof

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Embodiment 1

[0044] This embodiment is the preparation method (and amplification efficiency analysis) of Danshen EST-SSR molecular marker, and its preparation method includes the following main steps:

[0045] (1) Obtaining and preprocessing the EST sequence of Salvia miltiorrhiza

[0046] In December 2008, a total of 10288 Danshen EST sequences were downloaded from GenBank (http / / :www.ncbi.nlm.nih.gov / dbEST) in FASTA format. Use VecScreen and RepeatMasker to remove vector contamination and repetitive sequences, use EST-trimmer to remove polyT or polyA at the 5' or 3' end, use Phrap to perform EST contig analysis and clustering to remove redundant EST sequences, and remove ESTs with a length less than 100bp sequence. After sorting the Danshen EST sequences registered in GenBank, a total of 4192 non-redundant Uni-EST sequences with a total length of about 2026kb were obtained after splicing, including 1276 contigs and 2916 singletons.

[0047] (2) Screening of SSR sites in the EST sequenc...

Embodiment 2

[0065] This embodiment is that the primers obtained in Example 1 are used for genetic diversity analysis and germplasm identification of Salvia miltiorrhiza:

[0066] 1. Test materials

[0067] Thirteen samples of Salvia miltiorrhiza were selected, including 7 wild populations and 6 cultivated populations, and 10 closely related species of the genus Salvia were selected to detect the application of EST-SSR markers in Danshen in genetic diversity analysis and germplasm identification. Validity, for details, see figure 2 .

[0068] 2. DNA extraction

[0069]For each sample, 3 individual plants were randomly selected, young leaves were collected and mixed in equal amounts, and 0.2-0.5 g of fresh young leaves were ground into powder in liquid nitrogen; the powder was transferred to a 1.5 mL centrifuge tube, and 500 μL of preheated extraction buffer ( 2% CTAB, 100mM Tris-HCl, pH 8.5, 50mM EDTA, pH 8.0, 100mM NaCl), in a water bath at 65°C for 60min, and mix several times; add a...

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Abstract

The invention discloses a method for preparing a salvia miltiorrhiza EST-SSR molecular mark. The method mainly comprises the following steps of obtaining and pre-processing a salvia miltiorrhiza EST sequence, screening a SSR locus in the salvia miltiorrhiza EST sequence, designing and synthesizing the salvia miltiorrhiza EST-SSR primer, screening the salvia miltiorrhiza EST-SSR primer and the like. The method excavates the SSR locus aiming at the salvia miltiorrhiza EST and designs the PCR primer according to the flanking sequence of the SSR locus, and on the basis of this, the salvia specificEST-SSR mark system is established; and the method lays a solid foundation for genetic diversity evaluation, genetic relationship or idioplasmatic identification, genetic linkage description, molecular mark auxiliary selection, gene mapping cloning, functional genome analysis, related researches and the like of related species of the salvia miltiorrhiza and the salvia. The invention also discloses the EST-SSR locus specific primer obtained by the method for preparing the salvia miltiorrhiza EST-SSR molecular mark and the application thereof.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and molecular genetics research of Chinese medicinal materials, in particular to a preparation method of EST-SSR molecular markers of salvia miltiorrhiza, specific primers and applications thereof. Background technique [0002] Salvia miltiorrhiza is a famous medicine for promoting blood circulation and removing blood stasis. Salvia miltiorrhiza, a plant of the genus Salvia miltiorrhiza, recorded in the Pharmacopoeia of the People's Republic of China, is its only source plant. It is used as medicine with dried roots and rhizomes. It is one of the earliest and most widely used medicines in traditional Chinese medicine to treat angina pectoris and coronary heart disease. In recent years, the research on the chemical constituents and pharmacological effects of Danshen has become increasingly in-depth, but there are relatively few reports on its population genetic diversity, germplasm classific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/11C12Q1/68
Inventor 张勇邓科君熊丙全彭金华赵晓楠任正隆
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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