CN110205397B - Wushan epimedium and EST-SSR molecular identification method of easily-mixed species thereof - Google Patents

Abstract

The invention discloses an EST-SSR molecular identification method for epimedium wushanense and easily-mixed species thereof, belonging to molecular markers and medicinal material identification in the technical field of molecular biology. The EST-SSR primer group for identifying epimedium wushanense and easily-mixed species thereof comprises 10 pairs of EST-SSR core primers and universal M13 primers: SEQ ID NO: 1.2 … …, 21. The identification method comprises the following steps: (1) extracting total DNA of a sample by using a CTAB method; (2) carrying out PCR amplification by taking the total DNA of a sample to be detected as a template; (3) analyzing the PCR amplification product; (4) and constructing a phylogenetic tree. The application is to identify Wushan epimedium and easily-mixed species thereof. The 10 SSR molecular markers provided by the invention have good polymorphism, strong specificity and stable amplification result, can realize the rapid and accurate identification of epimedium wushanense and easily-mixed species thereof, provide technical support for the improvement of the quality standard of epimedium medicinal materials, and are favorable for the development and utilization of the epimedium medicinal materials.

Description

Wushan epimedium and EST-SSR molecular identification method of easily-mixed species thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to molecular markers and medicinal material identification, in particular to epimedium wushanense (Wushan epimedium) (Wushan)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping Epimedium (herba Epimedii)E. ilicifolium) Epimedium wushanense (epimedium wushanense) (wushu)E. pseudowushanense) Qianbei epimedium (Epimedium brevicornum Maxim.)E. borealiguizhouense) The EST-SSR molecule identification method.

Background

The herba Epimedii is dried leaf of plant of Epimedium of Berberidaceae (Berberidaceae). There are about 62 species of Epimedium Genus currently known in The world, and about 52 species in China (Stearn, WT. The Genus Epimedium and Other Herbacous Berberidaceaeand, timer Press, 2002; ying, TS., boufford, DE., brach, AR. Epimedium L. St. Louis, science Press, missouri national Garden Press, 2011, 787-799.), each version of Chinese pharmacopoeia includes 5 kinds of Epimedium, including Epimedium (Epimedium herb)Epimedium brevicornuMaxim), epimedium sagittatum (Maxim)E. sagittatumMaxim), epimedium pubescens (herba Epimedii), (herba Epimedii (Maxm.) Merr. (Maxmm)E. pubescensMaxim), epimedium koreanum (Epimedium koreanum nakai) ((III)E. koreanumNakai and Epimedium wushanense (Wushan)E. wushanense T.s.ying), 2015 edition of chinese pharmacopoeia listed wushanense alone as a species called wushanense. There are over ten species of epimedium plants used as epimedium medicinal materials in various local standards and folk traditions. As a traditional Chinese medicine in large circles, epimedium has been used for more than 2000 years in China, is one of The most widely applied Chinese herbal medicines with The most development potential, not only has The efficacies of tonifying kidney Yang, strengthening bones and muscles, dispelling wind-damp and The like, but also has various efficacies of resisting aging, improving immune function, protecting cardiovascular and cerebrovascular systems, resisting inflammation, resisting virus, relieving cough and asthma, inhibiting tumors, regulating liver lipid metabolism, improving Alzheimer disease and The like (Ma HP, he XR, yang Y, li MX, hao DJ, jia ZP., the genus Epimedium: an ethnopharmacological metabolic disease Journal of Ethylallopharmacological bearing, japan 5363, 2015134 (3), 519-541, jiang J, song J, jia XB. and Phytopharmacological binding and Ethopological disease (204), and The version of Chinese herbal medicines (2011, 20157-204, 2015 3, 2015-541.

Wushan epimedium, taken as a species recorded in Chinese pharmacopoeia, is more disordered in use because a plurality of species are similar to leaf shapes thereof and are all in a scalpeloid shape or a scalpeloid shape. Studies have shown that the epimedium wushanense described by Flora of China actually includes 4 species with similar leaf morphology and completely different flower morphology, except for epimedium wushanense, also includes epimedium zhenping (A)E. ilicifoliumStearn), jin Chengshan Epimedium (E. jinchengshanenseY.J.Zhang &J.Q.Li), epimedium wushanense (epimedium wushanense)E. pseudowushanenseB.L.Guo）(Zhang YJ, DangHS,LiJQ,Wang Y,The Epimedium wushanense (Berberidaceae) species complex, with one new speciesfrom Sichuan,China, phytotaxa,2014,172 (1): 039-045). Qianbei epimedium (A) in the recipeE. borealiguizhouenseS.Z.He &Y.k. Yang) has needles to needles shaped leaves, and is also commonly used as epimedium wushanense. Epimedium wushanense, epimedium zhengtarsonchii, epimedium Jin Chengshan and Epimedium wushanense belong to the large flower group, and the diameter of the flower is 3.5-4cm; the petals of Epimedium wushanense and Epimedium wushanense have long distance, and have petals away from the base, and are all ser.DavidianaeStearn, but yellow flowers of Epimedium wushanense with basal petals about 7mm high, and purple flowers of Epimedium wushanense with basal petals slightly about 2-3mm high; the petals of the ballast epimedium and Jin Chengshan epimedium have long distances and do not have basal petals, and both belong to ser.DolichoceraeStearn, but 5-6X 2.5mm for Zhenping epimedium inner sepal, and 10-12X 5-6mm for Jin Chengshan for epimedium inner sepal. Epimedium Qianbei belongs to the group of small flowers, with a diameter of about 5mm, and belongs to ser.BrachyceraeStearn, the petals are slipper-shaped and about 2.5mm long. Although the forms of the epimedium wushanense, the epimedium glabrata, the epimedium Jin Chengshan, the epimedium wushanense and the epimedium Qianbei have obvious differences in flowers, inflorescences and the like, the leaves of the application parts are highly similar and are often mixed in the use of medicinal materials, and the composition and the content of the medicinal components of different species have larger differences, so that the development of molecular markers capable of quickly identifying the epimedium wushanense and the easily mixed species thereof is urgently needed.

Disclosure of Invention

The invention aims to provide an EST-SSR molecular identification method of epimedium wushanense and easily-mixed species thereof, namely an effective method for quickly identifying epimedium wushanense and easily-mixed species thereof, namely, ballast epimedium Jin Chengshan, epimedium wushanense and epimedium Qianbei by utilizing SSR molecular marker combination.

The object of the invention is achieved by the following steps:

1. EST-SSR primer group for identifying epimedium wushanense and easily-mixed species thereof

The EST-SSR primer group for identifying the epimedium wushanense and the miscible species thereof comprises 10 pairs of EST-SSR core primers and universal M13 primers; wherein, the sequence of the universal M13 primer is 5'-GTAAAACGACGGGCCAGT-3', and the sequence information of 10 pairs of EST-SSR core primers is shown in Table 1:

TABLE 1 sequence information for EST-SSR core primers

2. Wushan epimedium and EST-SSR molecular identification method of easily-mixed species thereof

The method comprises the following steps:

(1) cleaning the surface of a leaf of a sample to be detected by using 75% alcohol, and extracting total DNA of the sample by using a CTAB method;

(2) taking total DNA of a sample to be determined as a template, and carrying out PCR by using an EST-SSR primer group, wherein the PCR reaction system is as follows: 5 mu L of 2 xTaq PCR Master Mix reaction buffer solution, 0.0125 mu L of 10 mu M upstream primer and 0.25 mu L of 10 mu M downstream primer, 0.15 mu L of M13-ROX/FAM/HEX/TAMRA joint primer, 3 mu L of diluted DNA template, and supplementing 10 mu L of sterile water; the PCR amplification procedure was: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing temperature from 65 deg.C to 50 deg.C for 15 cycles, reducing each cycle by 1 deg.C, performing 22 cycles at 50 deg.C, extending at 50 deg.C for 30s, extending at 72 deg.C for 42s, and storing at 16 deg.C;

(3) analyzing PCR amplification products, adding 1 mu L of 10 multiplied by DNA Loading Buffer into 9 mu L of PCR products, fully mixing, adding to a sample application space, adding 2 mu L of 2000 DNA Marker into an empty sample application hole, and observing the amplification condition by a full-automatic gel imaging analysis system after electrophoresis for 30 min; after observing the band of interest, the following operations were performed: respectively adding 90 mu L of sterile water into 10 mu L of PCR products with HEX, FAM, ROX and TAMARE fluorescent groups, taking 5 mu L of diluent from each plate to a sterile 96-well plate after full mixing, adding 80 mu L of sterile water, taking 10 mu L of mixed solution to 30 mu L of sterile water after full mixing, fully mixing 4 mu L of diluent to 6 mu L of HIDI solution, adding 1.8 mu L of LIZ500 internal standard into 1mL of HIDI solution before use, and detecting by an ABI3730 genetic analyzer after full mixing;

(4) and constructing a phylogenetic tree, reading the length of the allele fragment through a GeneMark 2.4.0, correcting the data in an Excel table after the data reading is finished, constructing a UPGMA tree through a PowerMark, and identifying the species group to which the sample to be detected belongs.

3. Application of Wushan epimedium and EST-SSR molecular identification method of easily-mixed species thereof

Epimedium wushanense (Wushan epimedium)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping epimedium (herba epimedii) ((herba epimedii))E. ilicifolium) Epimedium wushanense (epimedium wushanense) (wushu)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) And (5) performing identification.

Compared with the prior art, the invention has the following advantages and positive effects:

(1) the invention establishes epimedium wushanense (epimedium wushanense)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping Epimedium (herba Epimedii)E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) The EST-SSR molecule identification method of (1); the traditional morphological identification needs complete morphological characteristics including flowers, inflorescences and the like to accurately identify, and epimedium medicinal materials circulate leaves, so that although the leaf morphologies of epimedium wushanense and other 4 miscible species have slight differences, the identification difficulty is very high;

(2) the 10 SSR molecular markers provided by the invention have good polymorphism, strong specificity and stable amplification result, can realize the rapid and accurate identification of epimedium wushanense and easily-mixed species thereof, provide technical support for the improvement of the quality standard of epimedium medicinal materials, and are favorable for the development and utilization of the epimedium medicinal materials.

Drawings

FIG. 1 shows Epimedium wushanense (Wushan)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping epimedium (herba epimedii) ((herba epimedii))E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) Detecting the result of agarose electrophoresis of the sample DNA;

FIG. 2 shows EST-SSR primer C43 and general primer M13-HEX in Epimedium wushanense (II)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping epimedium (herba epimedii) ((herba epimedii))E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) After the PCR amplification product is detected by a genetic analyzer, a peak image is read by a GeneMarker 2.4.0;

FIG. 3 shows the primer sets based on Epimedium wushanense (Wushan)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping Epimedium (herba Epimedii)E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) And (3) typing data of PCR amplification products of each DNA sample to construct UPGMA trees.

English-Chinese translation

EST-SSR: ESTs (Expressed sequence tag) are Expressed sequence tags; SSR (Simple sequence repeat) is Simple sequence repeat; EST-SSR is an expression sequence label microsatellite, and is a novel molecular marker repeatedly developed based on a simple sequence of an expression sequence label.

Detailed Description

The invention will be further illustrated with reference to the following figures and specific examples without however limiting the scope of the invention.

Example 1: epimedium SSR primer design

Performing SSR search on Unigenes with more than 1kb of epimedium transcriptome in a database by using MicroSatellite (MISA) software according to the standards of 10, 6 and 5 repeated times of mononucleotide, dinucleotide and trinucleotide respectively; designing a target Primer for the searched SSR locus by using Primer 6 software, wherein the design standard is as follows: the length of the primer is 18-26bp, the annealing temperature is 50-65 ℃, the length of the PCR amplification final product is 100-350bp, and 5471 pairs of primers capable of being compared with NCBI non-redundant protein database are selected for SSR locus synthesis, wherein each pair of primers consists of an upstream primer F and a downstream primer R.

Example 2: extraction and detection of sample genomic DNA

The method comprises the following steps of extracting and detecting genome DNA of leaves of Wushan epimedium and 4 miscible species of Wushan epimedium, wherein the steps are as follows:

A. selecting tender leaves of a sample to be detected, removing surface dust by using 75% alcohol, preheating a CTAB buffer solution at 65 ℃, and precooling isopropanol at-20 ℃;

B. taking 0.1g of dried plant leaves, quickly freezing the plant leaves by liquid nitrogen, fully grinding the plant leaves into powder, subpackaging the powder into 2mL centrifuge tubes, adding 1mL of CTAB buffer solution preheated to 65 ℃, uniformly mixing the buffer solution by turning upside down, preserving the temperature at 65 ℃ for 30min, and shaking the mixture uniformly every 10 min;

C. after incubation, 12000r/min, centrifuging for 10min, taking 900 mu L of supernatant, adding chloroform-isoamylol (24;

D. repeating the step C, adding equal volume of isopropanol precooled at-20 ℃, shaking uniformly, settling at-20 ℃ for more than 30min, centrifuging at 12000r/min and 4 ℃ for 10min by using a precooling 4 ℃ centrifuge, and removing supernatant;

E. adding 1mL of 70% ethanol, reversing the mixture from top to bottom for several times until the precipitate floats, centrifuging the mixture at 12000r/min and 4 ℃ for 10min, and removing the supernatant;

F. e, repeating the step E, airing at room temperature in a fume hood, adding 60 mu L of TE to dissolve DNA, and storing at-20 ℃ for later use after dissolution;

G. assembling a comb and a gel plate, uniformly mixing 1.2g of agarose and 100mL of 1 XTAE, heating and dissolving the mixture in a microwave oven, cooling the mixture to about 45 ℃, adding 10 mu L of Gold View, uniformly mixing the mixture, and pouring the mixture into gel plate gel;

H. adding 3 mu L of DNA solution into 1 mu L of 6 XDNA Loading Buffer, uniformly mixing, adding into a sample application hole, performing 120V constant voltage electrophoresis for 30min, observing DNA bands by a full-automatic gel imaging analysis system, and taking the DNA bands with high brightness, clarity and no dispersion as qualified extracted DNA.

As shown in FIG. 1, is Epimedium wushanense (Wushan epimedium) (Wushan)E. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping epimedium (herba epimedii) ((herba epimedii))E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (Epimedium brevicornum Maxim.)E. borealiguizhouense) On the agarose gel electrophoresis image, clear and bright DNA bands were observed.

Example 3: PCR amplification

Carrying out PCR amplification on genomic DNA of epimedium wushanense and 4 miscible species thereof by using synthesized 10 pairs of EST-SSR core primers and universal M13 primers, wherein the reaction system is as follows: 5 mu L of 2 xTaq PCR Master Mix reaction buffer solution, 0.0125 mu L of 10 mu M upstream primer and 0.25 mu L of 10 mu M downstream primer, 0.15 mu L of M13-ROX/FAM/HEX/TAMRA joint primer, 3 mu L of diluted DNA template, and supplementing 10 mu L of sterile water; the PCR amplification procedure was: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing temperature from 65 ℃ to 50 ℃ for 15 cycles, reduction of 1 ℃ per cycle, annealing at 50 ℃ for 22 cycles, extension at 50 ℃ for 30s, final extension at 72 ℃ for 42s, and storage at 16 ℃.

Example 4: PCR amplification product detection

The method comprises the following steps of carrying out PCR amplification on genomic DNA of epimedium wushanense and 4 miscible species of the epimedium wushanense by utilizing synthesized 10 pairs of EST-SSR core primers and universal M13 primers, and detecting PCR products, wherein the specific method comprises the following steps:

adding 9 mu L of PCR product into 1 mu L of 10 multiplied DNA Loading Buffer, fully mixing, adding to a sample application space, adding 2 mu L of 2000 DNA Marker into the sample application hole, performing electrophoresis for 30min, and observing the amplification condition by a full-automatic gel imaging analysis system;

after observing the band of interest, the following operations were performed: respectively adding 90 mu L of sterile water into 10 mu L of PCR products with HEX, FAM, ROX and TAMARE fluorescent groups, taking 5 mu L of diluent from each plate after full mixing, adding 80 mu L of sterile water into a sterile 96-well plate, taking 10 mu L of mixed solution after full mixing, fully mixing in 30 mu L of sterile water, taking 4 mu L of diluent into 6 mu L of LHIDI solution, adding 1.8 mu L of LIZ500 internal standard into 1mL of HIDI solution before use, fully mixing, and detecting by an ABI3730 genetic analyzer.

Example 5: screening of SSR primers

The lengths of the reading allele fragments are read through GeneMarker 2.4.0, and SSR primers which are good in polymorphism, strong in specificity and stable in amplification result in epimedium wushanense and 4 miscible species samples are screened out.

FIG. 2 shows EST-SSR primer C43 and general primer M13-HEX in Epimedium wushanense (II)E. wushanense) And its easily-mixed species Jin ChengshanEpimedium (A) and (B)E. jinchengshanense) Zhenping Epimedium (herba Epimedii)E. ilicifolium) Epimedium wushanense (epimedium wushanense) (A)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) After the PCR amplification product is detected by a genetic analyzer, a peak image read by a GeneMarker 2.4.0 has better amplification stability and polymorphism.

Example 6: cluster analysis

The PCR product detection data of the primer group on epimedium wushanense and 4 miscible species thereof are analyzed by GeneMarker 2.4.0, and correcting in Excel table, and constructing UPGMA tree by Powermarker, as shown in FIG. 3, herba Epimedii (Uighur) of Wushan may be addedE. wushanense) And its miscible species Jin Chengshan Epimedium (E. jinchengshanense) Zhenping epimedium (herba epimedii) ((herba epimedii))E. ilicifolium) Epimedium wushanense (epimedium wushanense) (wushu)E. pseudowushanense) Qianbei epimedium (A), qianbei (B)E. borealiguizhouense) Effectively separated.

Sequence listing

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Claims (3)

1. An EST-SSR primer group for identifying epimedium wushanense and easily-mixed species thereof is characterized in that:

comprises 10 pairs of EST-SSR core primers and universal M13 primers;

the sequence of the primer C17 is shown as SEQ ID NO.1 and 2, the sequence of the primer C37 is shown as SEQ ID NO.3 and 4, the sequence of the primer C39 is shown as SEQ ID NO.5 and 6, the sequence of the primer C43 is shown as SEQ ID NO.7 and 8, the sequence of the primer C44 is shown as SEQ ID NO.9 and 10, the sequence of the primer C48 is shown as SEQ ID NO.11 and 12, the sequence of the primer C69 is shown as SEQ ID NO.13 and 14, the sequence of the primer C78 is shown as SEQ ID NO.15 and 16, the sequence of the primer C80 is shown as SEQ ID NO.17 and 18, and the sequence of the primer C90 is shown as SEQ ID NO.19 and 20;

the general M13 primer sequence is shown in SEQ ID NO. 21.

2. The EST-SSR molecular identification method of Wushan epimedium and miscible species thereof is characterized by comprising the following steps:

(1) cleaning the surface of a leaf of a sample to be detected by using 75% alcohol, and then extracting the total DNA of the sample by using a CTAB method;

(2) carrying out PCR by using total DNA of a sample to be determined as a template and the EST-SSR primer group for identifying epimedium wushanense and easily-mixed species thereof according to claim 1, wherein the PCR reaction system is as follows: 5 mu L of 2 xTaq PCR Master Mix reaction buffer solution, 0.0125 mu L of 10 mu M upstream primer and 0.25 mu L of 10 mu M downstream primer, 0.15 mu L of M13-ROX/FAM/HEX/TAMRA linker primer, 3 mu L of diluted DNA template and 10 mu L of sterile water; the PCR amplification procedure was: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, annealing temperature from 65 deg.C to 50 deg.C for 15 cycles, reducing each cycle by 1 deg.C, performing 22 cycles at 50 deg.C, extending at 50 deg.C for 30s, extending at 72 deg.C for 42s, and storing at 16 deg.C;

the Epimedium wushanense and its easily-mixed species include Epimedium Jin Chengshan, epimedium zhengchan, epimedium wushanense and Epimedium Qianbei;

(3) analyzing PCR amplification products, adding 1 mu L of 10 multiplied by DNA Loading Buffer into 9 mu L of PCR products, fully mixing, adding to a sample application space, adding 2 mu L of 2000 DNA Marker into an empty sample application hole, and observing the amplification condition by a full-automatic gel imaging analysis system after electrophoresis for 30 min; after observing the band of interest, the following operations were performed: respectively adding 90 mu L of sterile water into 10 mu L of PCR products with HEX, FAM, ROX and TAMARE fluorescent groups, taking 5 mu L of diluent from each plate to a sterile 96-well plate after fully mixing, adding 80 mu L of sterile water, taking 10 mu L of mixed solution to 30 mu L of sterile water after fully mixing, fully mixing 4 mu L of diluent to 6 mu L of HIDI solution, adding 1.8 mu L of LIZ500 internal standard into every 1mL before the HIDI solution is used, and detecting by an ABI3730 genetic analyzer after fully mixing;

(4) and constructing a phylogenetic tree, reading the length of the allele fragment through a GeneMark 2.4.0, correcting the data in an Excel table after the data reading is finished, constructing a UPGMA tree through a PowerMark, and identifying the species group to which the sample to be detected belongs.

3. The use of the method for identifying EST-SSR molecules in epimedium wushanense and its miscible species according to claim 2, wherein:

identifying Epimedium wushanense and its easily-mixed species Jin Chengshan Epimedium, epimedium zhengchangshan, epimedium wushanense and Epimedium Qianbei.

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