The acquisition method and application of the est-ssr marker primer set developed based on the transcriptome sequence of Ligusticum chuanxiong
A technology of transcriptome sequence and labeled primers, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve the problem of not utilizing Chuanxiong to transfer SSR-labeled primers, the difficulty of in-depth research on Chuanxiong molecules, and the limitations of Chuanxiong. Rapid and effective breeding and production application of high-quality germplasm resources, to achieve the effect of promoting molecular breeding and functional gene discovery of Rhizoma Chuanxiong, high polymorphism and accurate results
Inactive Publication Date: 2020-09-08
四川省农业科学院经济作物育种栽培研究所
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There are few reports on the genetic diversity of Rhizoma Chuanxiong, germplasm fingerprints, gene mapping, genetic map construction, molecular breeding, etc. There are few molecular markers in Rhizoma Chuanxiong that can be used for the above research, and they are some general primers, such as RAPD, ISSR , ITS, etc., making it difficult to carry out in-depth molecular research on Rhizoma Chuanxiong, which seriously limits the rapid and effective breeding and production application of high-quality germplasm resources of Rhizoma Chuanxiong.
However, there is no report on the use of Ligusticum chuanxiong to transfer SSR-labeled primers
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Embodiment 1
[0033] This embodiment prepares the Rhizoma Chuanxiong EST-SSR labeled primer set, comprising the following steps:
[0034] (1) Acquisition of SSR primers for transcriptome data of Rhizoma Chuanxiong
[0035] (1.1) Splicing the transcriptome sequencing data of Rhizoma Chuanxiong root, splicing and removing redundancy to obtain unigene.
[0036] (1.2) Use MISA software to mine the SSR sites of the spliced unigene. The basic parameters are set to repeat at least 10 times for single nucleotides, repeat at least 6 times for dinucleotides, repeat at least 5 times for trinucleotides, and repeat at least 5 times for tetranucleotides. Acids are repeated at least 5 times, pentanucleotides are repeated at least 5 times, and hexanucleotides are repeated at least 5 times.
[0037] (1.3) Use Primer 3.0 software to design primers for the above SSR detection results. The parameters are set as primer length 18-25bp; annealing temperature 52.0-60.0°C, and the Tm value difference between ups...
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The invention discloses an EST-SSR marker primer group developed on the basis of the sequence of a transcriptome of ligusticum wallichii, and an acquisition method and application of the EST-SSR marker primer group. The EST-SSR marker primer group developed on the basis of the sequence of the transcriptome of ligusticum wallichii comprises 74 pairs of primers, wherein the nucleotide sequences of the primers are shown as in SEQ ID NO:1 and SEQ ID NO:148 in a sequence table. The invention discloses the EST-SSR marker primer group of ligusticum wallichii for the first time, the primer is verified to obtain 74 pairs of EST-SSR with higher polymorphism, compared with a general primer, the EST-SSR marker primer group of ligusticum wallichii has the advantages of high polymorphism, easy amplification, high repeatability and more accurate results, can be used in the follow-up fundamental researches on construction of a genetic map and variety fingerprints of ligusticum wallichii, promotes the application researches on exploration of molecular breeding and functional genes of ligusticum wallichii and standardization of varieties of traditional Chinese medicines.
Description
technical field [0001] The invention belongs to the technical field of development and application of molecular marker technology, and more specifically relates to a method for obtaining and application of an EST-SSR marker primer set developed based on the sequence of the Chuanxiong transcriptome. Background technique [0002] Ligusticum chuanxiong is a medicinal plant of the genus Ligusticum umbelliferae. It is mainly planted in Pengzhou, Dujiangyan and other places in Sichuan. It is a famous traditional Chinese medicinal material in my country. Its dried rhizomes are used as medicine. Analgesic effect, used to treat headache, rheumatism, irregular menstruation, etc. [0003] The research on the active ingredients of Ligusticum Chuanxiong is very in-depth. Researchers have isolated many active ingredients including ligustrazine, ferulic acid, and ligustilide. The results of searching the research literature of Chuanxiong showed that there are many studies on the extraction...
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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 袁灿彭芳杨泽茂张超钟文娟杨晓牟方生龚一耘蒲德强戢沛城叶霄熊淼黄海燕
Owner 四川省农业科学院经济作物育种栽培研究所