阅读说明：本技术 基于棉花ssr分子标记筛选藏紫草的特异性ssr标记方法 (Specific SSR (simple sequence repeat) marking method for screening Tibetan lithospermum based on SSR molecular markers of cotton ) 是由 阿呷尔布 葛群 李俊文 谢红军 赵静 巩万奎 袁有禄 石玉真 商海红 潘境涛 邓 于 2018-12-19 设计创作，主要内容包括：本发明公开了一种基于棉花SSR分子标记筛选藏紫草的特异性SSR标记方法,其包括以下步骤：(1)提取藏紫草根皮部基因组DNA；(2)以第(1)步提取的基因组DNA为模板,选用特异性SSR引物进行PCR扩增；(3)对扩增产物进行凝胶电泳；(4)对电泳结果进行分析,其中所述特异性引物的碱基序列为：SHIN-1494F：TAACGGAGATGGGTTTCGAC；SHIN-1494R：CACAACCGTCCATTTCCTCT。本发明方法重复性好、多态性高、遗传稳定、不受环境条件影响,而且检测结果稳定可靠、快速准确、操作简单方便、成本低廉,有利于大规模的快速检测。(The invention discloses a specific SSR (simple sequence repeat) marking method for screening Tibetan lithospermum based on cotton SSR molecular markers, which comprises the following steps: (1) extracting the genomic DNA of the root bark of the Tibetan alkanna tinctoria; (2) taking the genomic DNA extracted in the step (1) as a template, and selecting a specific SSR primer for PCR amplification; (3) performing gel electrophoresis on the amplification product; (4) analyzing the electrophoresis result, wherein the base sequence of the specific primer is as follows: SHIN-1494F: TAACGGAGATGGGTTTCGAC, respectively; SHIN-1494R: CACAACCGTCCATTTCCTCT are provided. The method has the advantages of good repeatability, high polymorphism, stable heredity, no influence by environmental conditions, stable and reliable detection result, rapidness, accuracy, simple and convenient operation and low cost, and is beneficial to large-scale rapid detection.)

1. A method for identifying a specific SSR marker of Tibetan lithospermum is characterized by comprising the following steps:

(1) Extracting the genomic DNA of the Tibetan lithospermum to be detected;

(2) Taking the genomic DNA extracted in the step (1) as a template, and selecting a specific primer for PCR amplification;

(3) Analyzing the result, and if the amplified product simultaneously has two bands with molecular weights of 550bp and 235bp respectively, then the Tibetan lithospermum is the Onosma paniculatum;

Wherein the base sequence of the specific primer is as follows:

SHIN-1494F：TAACGGAGATGGGTTTCGAC

SHIN-1494R：CACAACCGTCCATTTCCTCT。

2. A specific SSR marker method according to claim 1 characterized in that: in the step (1), extracting the genomic DNA of the root bark of the Tibetan alkanna to be detected.

3. a specific SSR marker method according to claim 1 characterized in that: the analysis of the result is a gel electrophoresis analysis of the amplification product.

4. A specific SSR marker method according to claim 1 characterized in that: in the step (2), the PCR amplification reaction system is as follows: the reaction system is 10 mu 1, wherein the ultrapure water is 6.40 mu 1, 10 xBuffer 1.0 mu 1, 10mM dNTPs 0.50 mu 1, 10 mu M forward primer 0.50 mu 1, 10 mu M reverse primer 0.50 mu 1, 30 ng/. mu.1 template DNA1.0 mu 1, and 5U/. mu.1 Taq DNA polymerase 0.10 mu 1; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 45s for 1 cycle; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 1min, 29 cycles; denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 2min, one cycle.

5. A specific primer for identifying Tibetan lithospermum is characterized by comprising the following components in parts by weight:

The base sequence of the specific primer is as follows:

SHIN-1494F：TAACGGAGATGGGTTTCGAC

SHIN-1494R：CACAACCGTCCATTTCCTCT。

Technical Field

The invention relates to a specific SSR marker for screening Tibetan lithospermum based on cotton SSR molecular markers, which is used for carrying out molecular marker analysis and identification on different Tibetan lithospermum and belongs to the field of biotechnology application.

background

the Tibetan lithospermum is specially produced in Tibetan and belongs to the genus of Yunnan alkanna, has been used as a Tibetan medicine for thousands of years, is commonly used by Tibetan medicine for treating pneumonia, cavitary tuberculosis and high mountain hyperemia, is a main component of a common Tibetan medicine compound Tibetan medicine podophyllum lotion, Tibetan lithospermum dew, twenty-five-ingredient podophyllum pills, seven-ingredient blood disease pills and the like, and is also used as a good medicine for treating scalds and burns in folk. The Tibetan population also has the tradition of staining food with the juice of lithospermum erythrorhizon for a long time. However, the habitat of the lithospermum is wide,The variety is various, and most of the Yunnan alkannin plants are used as the Tibetan lithospermum for a long time in the Tibetan region. According to Chinese plant record, there are 6 kinds of Yunnan alkannis plants produced in Tibet. The regulation of the Chinese materia medica Tibetan medicated roll is as follows: the Tibetan radix Arnebiae is radix ArnebiaeOnosma hookeri Clarke, var, longiforum Duthie and Lithospermum erythrorhizonOnosma hookeriC.b. Clarke. However, through field investigation for many years and investigation by a plurality of Tibetan medicine experts, the morphology of the Tibetan medicine is closer to that of the common variety. The first-grade Tibetan alkannin has thin and thick roots, is longer than sand, is easy to pick and dig, has straight roots, does not have old skin mostly when being picked and dug, has basal leaves on the overground part, and has no more than 15 cm for the longest leaves. The four medical classics records: it is indicated for lung abscess, irregular menstruation and anemia by its root bark relieving cough and resolving phlegm. The record of "Jingzhu materia Medica": it has the actions of promoting blood circulation and treating lung diseases. Growing in sandy soil with little moisture, the roots are fine and juicy, and the quality is poor. The book Ruyi Baoshu says: clear lung heat, stop hematemesis. In the book of Tibetan medicine, the book of references: the medicine grows on the morning beach with hard upper quality, leaves are grey white, coarse, blue red flowers, red roots, sweet and slightly bitter, and has the effect of treating lung diseases, blood diseases and the like. The above book also only gives differences in quality between different habitat users. The reason for this difference is unknown whether the lithospermum-based source is a habitat. Previous studies by the present inventors have shown that commercially available Tibetan radix Arnebiae vary greatly in quality. At present, the molecular level research on the Tibetan lithospermum medicinal material is less, and further the molecular marker is utilized to have important significance for further research on the Tibetan lithospermum medicinal material.

SSR molecular markers, also called microsatellite markers, are tandem repeat DNA sequences consisting of 1-6 bases, widely exist in genomes of eukaryotes, and are mostly composed of relatively conserved single-copy sequences at two ends. The oligonucleotide motifs in the microsatellite sequence differ in the different genotypes of the same species and in the number of repeats and in the specific base sequence in different species. Compared with other markers, the SSR molecular marker has the characteristics of abundant quantity, abundant variation, low requirement on template DNA, co-dominant inheritance, good repeatability, strong coverage and the like. The SSR molecular markers are widely applied to genetic map construction of plants, QTL excavation, gene positioning, fingerprint map construction, variety identification, seed purity detection, species genetic diversity analysis and the like. At present, researchers successfully utilize the characteristic that DNA sequences between species have homology, and utilize developed SSR molecular markers or EST sequences and conserved repetitive DNA sequences in a public database to develop SSR molecular markers of species specificity, so that the problems of difficulty in developing markers of species without DNA sequence information, cost reduction, efficiency improvement and the like are solved, and the SSR molecular markers of cotton such as Zhenglisan and the like are utilized, and 111 pairs of effective polymorphic primers in different banana varieties are screened by adopting a transfer amplification method.

Cotton (A)Gossypiumspp.) is an important commercial crop, and a large number of SSR molecular markers have been developed and applied to cotton genetic map construction, QTL positioning, gene mining, fingerprint map construction, variety identification, seed purity detection and cotton genetic diversity analysis. The SSR molecular markers developed in the Tibetan lithospermum medicinal materials are very limited, and the genome information about the Tibetan lithospermum medicinal materials in a public database is very little, so that the inventor utilizes 55 pairs of cotton polymorphism SSRs primers to amplify the DNA of 42 different Tibetan lithospermum medicinal materials, screens out SSR primers capable of specifically amplifying in different Tibetan lithospermum medicinal materials, lays an important foundation for comparative genomics, variety identification, fingerprint map construction, QTL positioning of important characters, gene mining and molecular assisted selection breeding of the Tibetan lithospermum medicinal materials, and has important significance for research and application of the Tibetan lithospermum medicinal materials.

Disclosure of Invention

The invention aims to identify a specific SSR marker method of Tibetan lithospermum to overcome the defect of SSR molecular markers developed in Tibetan lithospermum medicinal materials.

The technical scheme provided by the invention is as follows: a specific SSR marker method for screening Onosma paniculatum on the basis of cotton SSR molecular markers comprises the following steps:

(1) Extracting the genomic DNA of the Tibetan lithospermum to be detected;

(2) Taking the genomic DNA extracted in the step (1) as a template, and selecting a specific primer for PCR amplification;

(3) Preferably, analyzing the amplification result by gel electrophoresis, and if the amplification product simultaneously has two bands with molecular weights of 550bp and 235bp respectively, then the Tibetan lithospermum erythrorhizon is the Onosma paniculatum;

Wherein the base sequence of the specific primer is as follows:

SHIN-1494F：TAACGGAGATGGGTTTCGAC

SHIN-1494R：CACAACCGTCCATTTCCTCT 。

The Tibetan lithospermum is used as a medicine from roots, and when medicinal materials with different basic sources are identified finally, in the step (1), the genomic DNA of the root bark of the Tibetan lithospermum is preferably extracted.

The specific SSR labeling method comprises the following steps of (2): the reaction system is 10 mu 1, wherein the ultrapure water is 6.40 mu 1, 10 xBuffer 1.0 mu 1, 10mM dNTPs 0.50 mu 1, 10 mu M forward primer 0.50 mu 1, 10 mu M reverse primer 0.50 mu 1, 30 ng/. mu.1 template DNA1.0 mu 1, and 5U/. mu.1 Taq DNA polymerase 0.10 mu 1; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 45s for 1 cycle; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, extension at 72 ℃ for 1min, 29 cycles; denaturation at 94 ℃ for 60s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 2min for one cycle; storing at 4 ℃ for later use.

The invention has the following beneficial effects: the invention discloses a specific SSR marker method for identifying Tibetan lithospermum based on research on screening the characteristic SSR marker of onosma paniculatum by cotton SSR molecular markers, which has the advantages of good repeatability, high polymorphism, stable heredity, no influence of environmental conditions and the like, and the detection result is stable, reliable, rapid and accurate, the operation is simple and convenient, the cost is low, and the large-scale rapid detection is facilitated.

The invention screens out a primer which can generate specific marker bands in different Tibetan lithospermum varieties and has clear band types, good repeatability and strong reliability from a large number of primers based on the research of screening the specific SSR markers of the Tibetan lithospermum by the cotton SSR molecular markers, and the primer is used as the specific primer for identifying the Pantoea paniculata. The specific primer SSR primer pair identifies different specific bands in different Tibetan lithospermum varieties. The SSR marker primer is stable in repeated expression, can be effectively used for identifying different varieties and types of the Tibetan lithospermum, can be verified mutually, lays an important foundation for further comparative genomics, variety identification, fingerprint map construction, QTL positioning of important characters, gene mining and molecular assisted selective breeding of the Tibetan lithospermum medicinal material, and has important significance for further research and application of the Tibetan lithospermum medicinal material.

the invention provides a specific SSR marking method for identifying Tibetan lithospermum, which has the advantages of rapidness, accuracy, economy, simplicity, convenience, stability, reliability, no influence of environmental conditions and development periods, simplicity in operation, convenience in statistics and the like, and can be used for rapidly and accurately detecting the genetic purity of cotton hybrid seeds.

Drawings

FIG. 1 is an SSR-PCR electropherogram of a primer for identifying detection characteristics of Tibetan radix Arnebiae, wherein M is Marker (D2000); the arrow indicates the specific banding pattern of the Onosma paniculatum; 1. 3, 4 is Yunnan arnebia euchroma (Royle) Johnst, 2, 8, 9, 10, 11 is Yunnan arnebia euchroma (Royle) Johnst, and 5, 6, 7 is Yunnan arnebia euchroma (Royle) Johnst.

Detailed Description

The invention is further illustrated by the SSR marker analysis examples based on PCR technology.