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Method for identifying salvia miltiorrhiza germplasm resource

A technology of Salvia miltiorrhiza and germplasm is applied to the development and application of SSR primers in the identification of plant germplasm resources, which can solve the problems of inability to contain the genetic information of Salvia miltiorrhiza, the application defects of universal resource identification of experimental programs, etc., and achieve accurate results and identification. Easy-to-follow, intuitive results

Inactive Publication Date: 2017-08-18
ZHEJIANG SCI-TECH UNIV
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Problems solved by technology

Although there have been cases of identifying germplasm resources of Danshen using SSR markers (Xu et al. Biochemical Systematics and Ecology 51(2013) 308–313; Wang Xueyong et al., Chinese Journal of Traditional Chinese Medicine, 2011, 36(3): 289-293) , but neither the source of the SSR markers nor the tested samples of Salvia miltiorrhiza (cultivated population, and not covering the distribution area of ​​Salvia miltiorrhiza) can contain relatively comprehensive genetic information of Salvia miltiorrhiza, and the versatility of the experimental program and the applicability of resource identification flawed

Method used

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  • Method for identifying salvia miltiorrhiza germplasm resource
  • Method for identifying salvia miltiorrhiza germplasm resource
  • Method for identifying salvia miltiorrhiza germplasm resource

Examples

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Embodiment 1

[0041] Example 1. Construction of Salvia miltiorrhiza transcriptome database

[0042] (1) Use the RNAprep Pure Plant Kit (TIANGEN, BEIJING) kit to extract total RNA from fresh leaves of Salvia miltiorrhiza, and send it to a sequencing company for high-throughput transcriptome sequencing.

[0043] (2) Use the De novo assembly function of GENEIOUS 9.0.2 to splice short-read sequences into transcriptome frame data, take the longest transcript in each gene as Unigene, and establish a transcriptome database capable of searching for microsatellite sites .

Embodiment 2

[0044] Example 2. Development of microsatellite SSR primers

[0045] (1) Use the MISA (http: / / pgrc.ipk-gatersleben.de / misa / misa.html) program package to scan, identify, and locate the different types of microsatellite sites in the Unigene above; parameter settings: identify single bases 1, 2, 3, 4, 5, and 6 base repeating units in the SSR region, and the corresponding repetition times should be at least 10, 6, 5, 4, 3, and 3 , and the base distance between two adjacent SSRs is at least 100bp;

[0046] (2) Copy *.fasta, misa.pl and misa.ini to the same folder, run the command >mias.pl*.fasta in the Perl environment, and get *.fasta.misa and *.fasta after running Two files of .statistics, among which *.fasta.misa is used for subsequent primer design.

[0047] (3) Use the web version of BatchPrimer 3 to design SSR primers, set parameters: primer length 18-27bp, annealing temperature (Tm) 55-65°C, product length 50-500bp; display according to *.fasta.statistics and *.fasta.misa ...

Embodiment 3

[0049] Example 3. Genomic DNA Extraction

[0050] Using DNA PlantZol reagent, the modified CTAB method was used to extract the genomic DNA of the above 222 Danshen materials, quantified by NanoDrop 2000, diluted to 20ng / μl, and stored at 4°C or -20°C until use.

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Abstract

The invention relates to a method for quickly identifying a salvia miltiorrhiza germplasm resource based on a salvia miltiorrhiza transcriptome sequence developed SSR primer label. The method mainly comprises the following steps: (1) collecting a wild salvia miltiorrhiza sample on the field, extracting DNA of all to-be-detected salvia miltiorrhiza population genomes, and detecting the extraction quality; (2) performing PCR amplification with a salvia miltiorrhiza transcriptome sequence-based SSR primer combination by taking the DNA extracted in the step (1) as a template; (3) performing sequence-based typing and gene segment length reading on the amplified product; and (4) establishing an SSR germplasm information feature library and germplasm resource identification framework of salvia miltiorrhiza by utilizing germplasm analysis software, and verifying the validity. The method can be used for effectively and quickly classifying and identifying wild salvia miltiorrhiza populations from different areas of China with correct and reliable results, is simple, convenient and feasible, has high applicability and strong repeatability, and can be effectively applied to germplasm resource identification of salvia miltiorrhiza.

Description

technical field [0001] The invention relates to the technical field of molecular markers, in particular to the development and application of SSR primers in the identification of plant germplasm resources. Through the method of transcriptome sequencing, 40 pairs of SSR primers with strong specificity and high polymorphism were efficiently developed. Using wild Danshen resources in 12 provinces of my country as materials, covering the natural distribution area and main production area of ​​the traditional Chinese medicine Salvia miltiorrhiza Established the SSR genetic information characteristic library of wild Danshen in my country and the identification framework of Danshen germplasm resources, and carried out the method of rapid germplasm resource identification. Background technique [0002] Salvia miltiorrhiza Bunge is one of the traditional bulk medicinal materials in China. Its natural groups are widely distributed in my country. It is a perennial herb of the Labiatae g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G06F19/24C12N15/11
CPCG16B40/00C12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113
Inventor 梁宗锁祁哲晨张晓丹王瑞红杨东风郭万里许玲沈超
Owner ZHEJIANG SCI-TECH UNIV
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