CN113684292A

CN113684292A - SSR molecular marker primer and method for identifying wild buckwheat in Guizhou region

Info

Publication number: CN113684292A
Application number: CN202011048796.XA
Authority: CN
Inventors: 王继永; 郑司浩; 李进瞳; 曾燕; 赵莎; 刘美娟; 尚兴朴; 王浩; 刘国城; 静一; 孙文静; 朱亚兵
Original assignee: China Traditional Chinese Medicine Seed & Seedling Co ltd; China National Traditional Chinese Medicine Co ltd; BGI Technology Solutions Co Ltd
Current assignee: China Traditional Chinese Medicine Seed & Seedling Co ltd; China National Traditional Chinese Medicine Co ltd; BGI Technology Solutions Co Ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-11-23
Anticipated expiration: 2040-09-29
Also published as: CN113684292B

Abstract

The invention provides an SSR molecular marker primer and a method for identifying wild buckwheat in Guizhou region, wherein the method comprises the following steps: carrying out PCR amplification by using the total DNA of the wild buckwheat rhizome sample to be identified as a template and an SSR molecular marker to obtain an amplification product; carrying out capillary electrophoresis sequencing typing on the amplification product to obtain a typing result of the amplification product; performing peak image interpretation on the typing result of the amplification product by using GeneMarker4.0 to obtain an allele matrix; calculating genetic diversity data of wild buckwheat rhizome by GenALEx according to the allele matrix; and (3) carrying out UPGMA clustering analysis by MEGA based on genetic diversity data obtained by GenALEx calculation to construct a clustering tree. The method solves the technical problems that the prior wild buckwheat rhizome producing area identification method mostly applies character identification, microscopic identification and the like, relies on personal experience and subjective judgment, has low accuracy and weak technical popularization.

Description

SSR molecular marker primer and method for identifying wild buckwheat in Guizhou region

Technical Field

The invention belongs to the field of molecular biological molecular markers, and particularly relates to SSR molecular marker primers and a method for identifying wild buckwheat in Guizhou region.

Background

The rhizoma Fagopyri Dibotryis is dry rhizome of Fagopyrum Dibotryis of Polygonaceae, and is called wild buckwheat, rhizoma Fagopyri Dibotryis, KAIJINKUO, and wild buckwheat root, and has effects of clearing heat and detoxicating, dispelling pathogenic wind and eliminating phlegm, promoting blood circulation and removing blood stasis, invigorating spleen and promoting diuresis, and can be used for treating sore throat, cough due to lung heat, lung carbuncle, phlegm odor, rheumatalgia, lung abscess, and dysentery.

The quality difference of the drug effect of wild buckwheat rhizome in different producing areas is large. The genuine property of the medicinal materials is that the medicinal effects of the medicinal materials are different due to the difference of ecological environments in various regions, and the medicinal effects can be better exerted only by the specific medicinal materials planted in specific geographical climates, and the genuine medicinal materials are essence of the genuine medicinal materials.

The identification of the wild buckwheat rhizome producing area is always a technical difficulty in identifying wild buckwheat rhizome species and is also an industrial problem faced by the traditional Chinese medicine resource industry at present. At present, the wild buckwheat production place identification mostly uses methods such as character identification and microscopic identification, and the like, and depends on personal experience and subjective judgment, so that the accuracy is not high, and the technology is not strong in popularization.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an SSR molecular marker primer and a method for identifying wild buckwheat in Guizhou region, and solves the technical problems that the existing wild buckwheat production place identification method mostly uses character identification, microscopic identification and other methods, depends on personal experience and subjective judgment, is low in accuracy and is not high in technology popularization.

The invention is realized by the following technical scheme:

an SSR molecular marker primer for identifying wild buckwheat in Guizhou region comprises the following primer pairs:

further, the annealing temperature of the primer pair is between 59 and 63 ℃, the length of the primer pair is between 21bp and 25bp, and the length of a PCR product is between 100bp and 200 bp.

Further, the method for identifying wild buckwheat in Guizhou region by using the SSR molecular marker primer comprises the following steps:

performing PCR amplification by using the SSR molecular marker primer by using the total DNA of the wild buckwheat rhizome sample to be identified as a template to obtain a PCR amplification product;

carrying out capillary electrophoresis sequencing typing on the PCR amplification product to obtain a typing result of the PCR amplification product;

performing peak plot interpretation on the typing result of the PCR amplification product by using GeneMarker4.0, and recording the size of an allele fragment to obtain an allele matrix;

calculating genetic diversity data of wild buckwheat among each population and each individual through GenALEx according to the allele matrix;

and (3) carrying out UPGMA clustering analysis by MEGA based on genetic diversity data obtained by GenALEx calculation to construct a clustering tree.

Further, extracting the total DNA of the wild buckwheat rhizome sample to be identified by using an mCTAB method.

Further, before the total DNA of the wild buckwheat rhizome sample to be identified is taken as a template, the purification of the total DNA of the wild buckwheat rhizome sample to be identified is also included.

Further, the reaction procedure of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 0.5min, annealing at 72 deg.C for 0.5min, extension at 72 deg.C for 1min, and repeating 32 cycles; extension at 72 ℃ for 10 min.

Further, the reaction system of the PCR amplification is as follows: ddH2O 11.8.8 μ L, 10 XTaq Buffer 2 μ L, 2mM dNTP 2 μ L, 5uM Forward primer 1 μ L, 5uM Reverse primer 1 μ L, Taq DNA polymerase 0.2 μ L, 50 ng/. mu.L of DNA template 2 μ L.

Further, before performing capillary electrophoresis sequencing and typing on the PCR amplification product, the method also comprises the step of performing electrophoresis detection on the PCR amplification product by using 1% agarose gel.

Further, the recording of the size of the allele fragment to obtain the allele matrix specifically includes: allele fragment sizes were recorded in a binary manner, i.e., 1 in the presence of an allele, 0 in the absence of an allele, and-9 in the absence of an allele, to obtain an allele matrix.

Further, the genetic diversity data includes a genetic similarity coefficient, an allelic factor NA, an expected heterozygosity HE, an observed heterozygosity HO, Shannon's index.

Compared with the closest prior art, the technical scheme of the invention has the following beneficial effects:

the invention applies genome technology and SSR molecular marker technology, utilizes wild buckwheat rhizome genome to design SSR primers, screens out 4 pairs of SSR molecular marker primers by wild buckwheat rhizome samples in Yunnan, Guizhou, Sichuan and the like, is used for identifying the wild buckwheat rhizome samples in the Guizhou area in a specific way, and has high accuracy, strong stability and easy large-scale popularization and application.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a UPGMA clustering chart of wild buckwheat rhizome samples in different main production areas constructed based on SSR molecular marker primers of the invention.

FIG. 2 is a schematic diagram of the identification result of an unknown sample of wild buckwheat rhizome based on the SSR molecular marker primer of the invention in a UPGMA cluster map.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment provides a method for identifying wild buckwheat in Guizhou region by SSR molecular marker primers, which specifically comprises the following steps:

s1: the method for extracting the total DNA of the wild buckwheat rhizome sample leaves to be identified by using the mCTAB method specifically comprises the following steps:

(1) putting 100mg of wild buckwheat rhizome sample leaves into a 2.0mL centrifuge tube, adding a small amount of quartz sand and a magnetic bead, immersing the wild buckwheat rhizome sample leaves into liquid nitrogen, freezing for about 1min, and grinding in a grinder to fine powdery materials for later use;

(2) adding 1.5mL buffer A (0.1M Tris-HCl pH8.0; 5mM EDTA; 0.25M NaCl; 1% PVP-40) solution into the ground powdery material, repeatedly inverting the centrifuge tube to homogenize and mix the solution and the material powder, performing ice bath for 10min, during which time, homogenizing and mixing the precipitated powder twice again, centrifuging at 10000 Xg, and removing the supernatant to obtain the first precipitate.

(3) Adding 1.5mL buffer A (0.1M Tris-HCl pH8.0; 5mM EDTA; 0.25M NaCl; 1% PVP-40) solution into the first precipitate, repeatedly inverting the centrifuge tube to homogenize and mix the solution and the material powder, performing ice bath for 10min, re-homogenizing and mixing the precipitated powder twice, centrifuging at 10000 Xg, and discarding the supernatant to obtain a second precipitate.

(4) To the second precipitate, 800. mu.L of a preheated 2% CTAB (0.1M Tris-HCl pH 8.0; 1.4M NaCl; 25mM EDTA; 2% (w/v) CTAB; 0.2% (v/v) beta-mercaptoethanol; 1% PVP-40) solution was added, and the precipitate was suspended homogeneously in the solution and stored in a 65 ℃ water bath for 1.5-2 hours while inverting the homogeneous solution 3-5 times to obtain a first mixed solution.

(5) The first mixture was centrifuged at 10000 × g at room temperature to obtain a first DNA supernatant, which was carefully poured into a new 2.0mL centrifuge tube, and an equal volume of CI (chloroform: isoamyl alcohol ═ 24:1(v/v)) solution was added thereto and mixed on an inverted shaker for 10min to obtain a second mixture.

(6) And centrifuging the second mixed solution by 10000 Xg to obtain a second DNA supernatant, carefully absorbing the second DNA supernatant into a new 2.0mL centrifuge tube by using a pipette gun, adding an equal volume of CI solution, and mixing for 10min on an inverted shaker to obtain a third mixed solution.

(7) Centrifuging the third mixed solution by 10000 Xg to obtain a third DNA supernatant, carefully sucking the third DNA supernatant into a new 1.5mL centrifuge tube by using a pipette gun, adding 0.6 volume times of ice-cold isopropanol, reversely mixing, and storing in a refrigerator at-20 ℃ for more than 1 hour to obtain a fourth mixed solution.

(8) And taking the centrifuge tube containing the fourth mixed solution out of the refrigerator, centrifuging by 10000 Xg, discarding the supernatant to obtain a first solidified body, pouring the centrifuge tube containing the first solidified body on dry paper to control the liquid drops to be dry as much as possible, adding 100 mu L of RNase solution (100mg/L), and storing at 37 ℃ for 0.5h to obtain a fifth mixed solution.

(9) Sequentially adding 150 mu L ddH into a centrifuge tube containing the fifth mixed solution₂And O, fully mixing 50 mu L of 5M NaCl and 700 mu L of absolute ethyl alcohol, centrifuging at 10000 Xg, discarding the supernatant to obtain a second solidified body, pouring the centrifuge tube containing the second solidified body on dry paper again, draining the liquid drops to the greatest extent, adding 600 mu L of 70% ethanol, mixing with a bullet bottom, centrifuging, discarding the supernatant to obtain a third solidified body.

(10) And adding 600 mu L of 70% ethanol into the centrifugal tube containing the third coagulation body again, mixing the mixture with the elastic bottom, centrifuging the mixture, and removing the supernatant to obtain the fourth coagulation body.

(11) And (3) drying the centrifugal tube containing the fourth coagulation body in vacuum to remove ethanol, and adding 100 mu LTE for dissolving to obtain a DNA dissolving solution.

The golden buckwheat rhizome sample acquisition information is as follows: the method comprises the following steps of respectively collecting 12, 10 and 9 Fagopyrum cymosum in Yunnan, Guizhou and Sichuan areas, wherein the specific information is shown in the following table 1:

TABLE 1

S2: purifying the total DNA of the wild buckwheat rhizome sample leaves to be identified, specifically, operating by using a gold hectorite agarose gel purification recovery kit (cargo number: DC011), and comprising the following steps:

(1) dropping the DNA solution into 1.5% agarose gel pores, and carrying out electrophoresis at 140V for 25min to obtain DNA bands.

(2) The DNA band to be purified and recovered was cut off with a clean blade under a long-wave ultraviolet lamp, and the gel containing no DNA was excised as much as possible.

(3) Putting the cut gel containing the DNA strips into a 1.5ml centrifuge tube, adding a sol solution with 3 times of volume (the sol solution is a reagent in a kit product), and placing in a water bath at 56 ℃ for 10 minutes (or until the gel is completely dissolved); vortex every 2-3 minutes to help accelerate dissolution and obtain a solution.

(4) Adding the obtained solution into an adsorption column EC (placing the adsorption column into a collection tube), standing at room temperature for 1 minute, centrifuging at 12000rpm for 30-60 seconds, and pouring off the waste liquid in the collection tube; then 700 mul of rinsing liquid WB is added, centrifugation is carried out at 12000rpm for 30 seconds, and waste liquid in a collecting pipe is discarded; then adding 500 mul of rinsing liquid WB, centrifuging at 12000rpm for 30 seconds, discarding the waste liquid in the collecting tube, and obtaining the adsorption column for adsorbing DNA.

(5) Putting the adsorption column adsorbing the DNA back into an empty collecting tube, centrifuging at 12000rpm for 2 minutes, discarding waste liquid in the collecting tube to remove rinsing liquid as much as possible so as to prevent ethanol remained in the rinsing liquid from inhibiting downstream reaction, taking the adsorption column adsorbing the DNA out of the collecting tube, putting the adsorption column into a clean centrifugal tube, adding 50 mu l of elution buffer EB (the elution buffer is better heated in a water bath at 65-70 ℃ in advance) into the middle part of an adsorption film of the adsorption column, standing at room temperature for 2 minutes, centrifuging at 12000rpm for 1 minute to obtain a purified DNA solution in the centrifugal tube, carrying out Nanodrop DNA concentration detection on the obtained purified DNA solution, diluting to 50 ng/mu l, and using the DNA solution for subsequent PCR amplification.

S3: primer selection

And selecting 24 pairs of primers with the base number of the repeating unit (base sequence) of 2bp-5bp and the repeating unit (base sequence) repeated for 5-10 times from SSR candidate primers screened based on the whole genome high-throughput data for carrying out a preliminary experiment. The length of the selected primer is between 20bp and 25bp, the theoretical annealing temperature is about 55 ℃, and the length of the PCR product is between 150bp and 350 bp.

8 wild buckwheat rhizome samples are selected for carrying out preliminary experiments, primers are selected from two dimensions of amplification success rate and polymorphism (capillary fluorescence electrophoresis mode), 4 pairs of SSR molecular marker primers with good polymorphism and high amplification success rate are finally selected, and when the primers are used for subsequent PCR amplification, the primer pairs (JQM04, JQM07, JQM19 and JQM21) of the selected SSR molecular marker primers are shown in the following table 2:

TABLE 2

In Table 2, F represents a forward primer, and R represents a reverse primer.

S4: taking the total DNA of the purified wild buckwheat rhizome sample leaves to be identified as a template, and carrying out PCR amplification by using the primer pair of the SSR molecular marker primer to obtain a PCR amplification product, wherein the PCR amplification process specifically comprises the following steps:

reaction system: 11.8. mu.L of double distilled water (ddH2O), 2. mu.L of 10 XTaq Buffer, 2. mu.L of 2mM deoxyribonucleoside triphosphate (dNTP), 1. mu.L of 5uM Forward primer (Forward primer), 1. mu.L of 5uM Reverse primer (Reverse primer), 0.2. mu.L of Taq DNA polymerase (Taq DNA polymerase), 2. mu.L of 50 ng/. mu.L DNA template.

Reaction procedure: pre-denaturation at 94 ℃ for 4 min; 32 cycles of three-step PCR (denaturation at 94 ℃ for 0.5min, annealing time 0.5min, annealing temperatures of JQM03 and JQM04 at 56 ℃, JQM07 and JQM21 at 58 ℃, JQM19 at 54 ℃ and extension at 72 ℃ for 1 min); extension at 72 ℃ for 10 min.

S5: the PCR amplification product was detected by electrophoresis on a 1% agarose gel at 140V for 15 minutes, and the gel was illuminated with a long-wave UV lamp to see if there was a band and the fragment size was appropriate to confirm the success of the experiment.

S6: the PCR amplification products are mixed according to equal proportion and sent to the Min-8 company of biological engineering (Shanghai) to be subjected to capillary electrophoresis detection and typing, so as to obtain the typing result of the PCR amplification products.

S7: performing peak pattern interpretation on the typing result of the PCR amplification product by using GeneMarker4.0, and recording the size of an allele fragment to obtain an allele matrix, wherein the method specifically comprises the following steps: allele fragment sizes were recorded in a binary manner, i.e., 1 in the presence of an allele, 0 in the absence of an allele, and-9 in the absence of an allele, to obtain an allele matrix.

S8: genetic diversity data of wild buckwheat between each intermediate population and each individual is calculated by GenALEx according to the allele matrix, and the genetic diversity data comprises but is not limited to genetic similarity coefficient, allele factor NA, expected heterozygosity HE, observed heterozygosity HO and Shannon's index.

The genetic diversity parameters for each population are shown in table 3 below:

TABLE 3

Where Pop is english output, indicating population, Na ═ observed allele factor, Ne ═ effective allele factor, I ═ Shannon's index, Ho ═ observed heterozygosity, He ═ desired heterozygosity, and F ═ 1- (Ho/He).

As can be seen from table 3:

the 3 population effective alleles were 2.650-3.590, and the Shannon's index was 1.189-1.499, with the Guizhou population having the lowest mean allele and the corresponding lower Shannon's index. The observation heterozygosity of the Yunnan and Sichuan communities is less than the expected heterozygosity, which indicates that the communities may have certain inbreeding phenomena or have the situation that heterozygosity loss is not detected; the observed heterozygosity of the Guizhou community is larger than the expected heterozygosity, which indicates that the community has a certain hybrid selection phenomenon or outcrossing phenomenon.

The genetic similarity coefficients between the various populations are shown in table 4 below:

TABLE 4

	Yunnan province	Guizhou province	Sichuan
				Yunnan province	1.000
Guizhou province	0.644	1.000
				Sichuan	0.413	0.308	1.000

As can be seen from table 4:

the average genetic similarity coefficient between the communities is 0.455, wherein the genetic similarity coefficient between Yunnan and Guizhou of the community is 0.644 at most, which indicates that the community relations between Yunnan and Guizhou communities are close, the genetic similarity coefficients between Sichuan and Yunnan and Guizhou of the community are 0.413 and 0.308 respectively, and indicates that the community relations between Sichuan and Yunnan and Guizhou community are relatively farther.

S9: based on the genetic diversity data calculated by GenALEx, UPGMA clustering analysis was performed by MEGA to construct a clustering tree, as shown in fig. 1.

As can be seen from FIG. 1, only one Guizhou population is relatively pure, and the other branches are mixed to different degrees, which also indicates that 4 pairs of SSR primers can better distinguish and identify the Guizhou gold buckwheat population. According to the results of the polymorphic sites of the 4 pairs of SSR primers and based on the genetic distance, if the wild buckwheat rhizome and the Guizhou are clustered together, the wild buckwheat rhizome production place is Guizhou.

The application case is as follows:

2 parts of wild buckwheat material (Sample1,2) with unknown origin are obtained from the market, the origin of Sample2 is Guizhou according to the identification result of the 4 pairs of SSR primer combinations, and the identification result is shown in FIG. 2.

In addition, FIGS. 1 and 2

The genetic distance scale of the dendrogram is shown, and the length of the line above 0.6 represents 0.6 genetic distances.

Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can make modifications and equivalents to the embodiments of the present invention without departing from the spirit and scope of the present invention, which is set forth in the claims of the present application.

Sequence listing

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BGI TECH SOLUTIONS Co.,Ltd.

CHINA TRADITIONAL CHINESE MEDICINE SEED & SEEDLING Co.,Ltd.

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Claims

1. An SSR molecular marker primer for identifying wild buckwheat in Guizhou region is characterized by comprising the following primer pairs:

2. an SSR molecular marker primer for identifying wild buckwheat in Guizhou region according to claim 1, wherein the annealing temperature of the primer pair is 59-63 ℃, the length of the primer pair is 21bp-25bp, and the length of a PCR product is 100bp-200 bp.

3. A method for identifying wild buckwheat rhizome in the Guizhou region by using the SSR molecular marker primers according to any one of claims 1 to 2, which comprises the following steps:

4. The method for identifying wild buckwheat rhizome in Guizhou region by SSR molecular marker primers according to claim 3, wherein the total DNA of the wild buckwheat rhizome sample to be identified is extracted by using an mCTAB method.

5. The method for identifying wild buckwheat rhizome according to claim 1 or 3, wherein the step of purifying the total DNA of the wild buckwheat rhizome sample to be identified is performed before the step of using the total DNA of the wild buckwheat rhizome sample to be identified as a template.

6. The method for identifying wild buckwheat rhizome in Guizhou region by SSR molecular marker primers according to claim 3, wherein the reaction procedure of PCR amplification is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 0.5min, annealing at 72 deg.C for 0.5min, extension at 72 deg.C for 1min, and repeating 32 cycles; extension at 72 ℃ for 10 min.

7. The method for identifying wild buckwheat rhizome in Guizhou region by SSR molecular marker primers according to claim 3, wherein the reaction system of PCR amplification is as follows: ddH2O 11.8.8 μ L, 10 XTaq Buffer 2 μ L, 2mM dNTP 2 μ L, 5uM Forward primer 1 μ L, 5uM Reverse primer 1 μ L, Taq DNA polymerase 0.2 μ L, 50 ng/. mu.L of DNA template 2 μ L.

8. The method for identifying wild buckwheat rhizome in Guizhou region by SSR molecular marker primers according to claim 3, wherein before the capillary electrophoresis sequencing and typing of the PCR amplification product, the method further comprises the step of detecting the PCR amplification product by electrophoresis with 1% agarose gel.

9. The method for identifying wild buckwheat in Guizhou region by SSR molecular marker primers according to claim 3, wherein the recording of the size of the allele fragments to obtain the allele matrix specifically comprises: allele fragment sizes were recorded in a binary manner, i.e., 1 in the presence of an allele, 0 in the absence of an allele, and-9 in the absence of an allele, to obtain an allele matrix.

10. The method for identifying wild buckwheat in Guizhou region by SSR molecular marker primers according to claim 3, wherein the genetic diversity data comprises genetic similarity coefficient, allelic factor NA, expected heterozygosity HE, observed heterozygosity HO, Shannon's index.