CN110438215B - Method for identifying affine plant variety by using ITS2 sequence - Google Patents
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Abstract
The invention discloses a method for identifying a affine plant variety by utilizing an ITS2 sequence, which comprises the following steps of 1, extracting DNA of a plant sample leaf to be identified; step 2, carrying out PCR amplification of ITS2 nucleotide sequences on DNA of leaves of the plant sample to be identified; step 3, carrying out bidirectional sequencing on the PCR product to obtain a leaf ITS2 spacer sequence of a plant sample to be identified; step 4, obtaining rDNA ITS2 sequences of homologous sequences and outer edge seed sequences; and 5, calculating the K2P genetic distance, constructing a phylogenetic tree, and identifying the Gnaphalium plant variety. The method for identifying the cudweed plant variety by utilizing the ITS2 sequence provides a molecular identification basis for identifying cudweed medicinal materials, can accurately and rapidly detect cudweed and easily-mixed products thereof from a plurality of traditional Chinese medicinal materials, improves the detection accuracy, and is suitable for wide popularization and application.
Description
Technical Field
The invention belongs to the technical field of biological identification methods, and particularly relates to a method for identifying a cudweed plant variety by using an ITS2 sequence.
Background
Gnaphalium (L.) belonging to the phylum Mortierella, class dicotyledonous, family Compositae, mostly annual herbs, and rarely perennial herbs, stems being grassy or basal slightly woody, dense white cotton wool or fluff; the leaves are intergrown leaves, all-edge leaves, and have no or short handles; the inflorescence is a head inflorescence, takes a shape of a poly umbrella or a developed cone, the total bud takes an oval shape or a bell shape, the total bud is golden yellow, faint yellow or yellow brown, a few of the total buds are reddish brown, 2-4 or 5 layers of the total buds are arranged, the back surface of the total buds are cotton wool, and the corolla is yellow or faint yellow; and (5) thinning fruits.
The Chinese plant Saint is recorded that the Gnaphalium plants are nearly 200 kinds worldwide, 19 kinds of Gnaphalium plants are available in China, the Gnaphalium plants are various, the Gnaphalium plants are similar in appearance and form and are difficult to distinguish, and the Gnaphalium plants are used as the pulsatilla in partial areas, and the reason is that: one is related to the color of the affine cudweed plant; secondly, the phenomenon of alien foreign matters exists in some places, and the affine cudweed is called a pulsatilla root; thirdly, the identification of the basic source, the character and the like requires professional identification personnel and is greatly influenced by subjective consciousness of people, so that a simpler and more accurate method is needed for carrying out the identification of the species.
The DNA barcoding (DNA barcoding) technology is used for rapidly and accurately identifying and identifying species by comparing a section of universal DNA fragment, has the characteristics of high efficiency, rapidness, high accuracy and the like, and is one of the main methods for identifying animal and plant medicinal materials in recent years. ITS2 is a non-coding region between rRNA coding genes 5.8S and 28S, is a moderately conserved sequence and is not long in length, has higher conservation in species, and has higher evolution rate and variation of different degrees among different species. The invention carries out molecular identification through the ITS2 sequence of the Gnaphalium medicinal plant, calculates the genetic relationship between the seed and the seed, and lays a foundation for research and further development and utilization of the Gnaphalium medicinal plant.
Disclosure of Invention
The invention aims to provide a method for identifying a cudweed plant variety by utilizing an ITS2 sequence, which solves the problem of low accuracy of the existing cudweed identification method.
The technical scheme adopted by the invention is that the method for identifying the cudweed plant variety by utilizing the ITS2 sequence comprises the following steps:
step 1, taking a plurality of leaves of a plant sample to be identified, and extracting DNA of the leaves of the plant sample to be identified;
step 2, carrying out PCR amplification of the ITS2 nucleotide sequence on the DNA of the plant sample leaf to be identified in the step 1 to obtain a PCR product;
step 3, performing bidirectional sequencing on the PCR product obtained in the step 2 through a sequencer to obtain a sequencing result, splicing and checking the sequencing result through Seqman software, removing 5.8S and 28S sections at two ends through hidden Markov model annotation, and obtaining and storing the ITS2 spacer sequence of the plant sample leaf to be identified;
wherein the gene coding of the leaf ITS2 spacer sequence of the plant sample to be identified is shown as a sequence 1;
step 4, based on the leaf ITS2 spacer sequence of the plant sample to be identified obtained in the step 3, performing Blast sequence comparison in an NCBI database to obtain a homologous sequence and an outer edge seed sequence of the Gnaphalium plant, and obtaining rDNA ITS2 sequences of the homologous sequence and the outer edge seed sequence;
and 5, calculating K2P genetic distance according to the ITS2 sequences obtained in the step 3 and the step 4, constructing a phylogenetic tree, and identifying the Gnaphalium plant variety.
The present invention is also characterized in that,
the step 1 specifically comprises the following steps:
step 1.1, taking a plurality of plant sample leaves to be identified, respectively placing the plant sample leaves into a centrifuge tube with the volume of 1.5mL, placing the centrifuge tube into liquid nitrogen for quick freezing, then taking out the plant sample leaves to be identified, placing the plant sample leaves into a plastic foam container containing the liquid nitrogen, and grinding the plant sample leaves into powder until the plant sample leaves are restored to room temperature;
step 1.2, preheating CTAB solution with the concentration of 2% (m/V) to 65 ℃ in a water bath, taking 700ul of the CTAB solution, adding the 700ul of CTAB solution into the plant sample leaves to be identified treated in the step 1, heating the plant sample leaves in the water bath at 65 ℃ for 30min, taking the plant sample leaves out, and placing the plant sample leaves in a room temperature environment;
step 1.3, after the solution treated in the step 1.2 is restored to room temperature, 450ul of phenol chloroform is added, vortex shaking is carried out twice under the condition of 14000rpm for 10min each time, standing is carried out, 420ul of upper liquid is sucked, the upper liquid is placed into a centrifuge tube with the volume of 1.5mL, then chloroform with the same volume is added, vortex centrifugal shaking is carried out for 10min under the condition of 14000rpm, standing is carried out, layering is carried out, and supernatant is taken;
step 1.4, taking 300ul of the supernatant in the step 1.3, adding isopropanol precooled to the temperature of minus 20 ℃ in an equal volume, standing for not less than 10min at the temperature of minus 20 ℃, centrifuging for 10min at 14000rpm, standing, and discarding the supernatant to obtain a white precipitate at the bottom of the tube;
step 1.5, adding 500ul of the white precipitate obtained in step 1.4, which has been pre-cooled to a volume fraction of-20 ℃ 70% ethanol, suspending and precipitating, shaking at 14000rpm for 2min, standing at room temperature for 10min, discarding supernatant, and adding 50ul ddH 2 And O, centrifuging for 1min at 14000rpm, and preserving at the temperature of 4 ℃ to obtain the leaf DNA of the plant sample to be identified.
The reaction system of the PCR amplification in the step 2 is as follows:
2 XTaq PCR Mix 12.5ul, and the common primers ITS2F and ITS3R each had a concentration of 10umol/L each of 1ul, and the DNA obtained in step 1 was 1ul and ddH2O 9.5ul.
The reaction conditions for PCR amplification in step 2 are:
denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 45s,40 cycles; the temperature was 72℃for 2min.
The PCR amplification primers in the step 2 are as follows:
ITS2F：5’-ATCGATGCGATACTTGGTGTGAAT-3’，
ITS3R：5’-ATCGGACGCTTCTCCAGACTACAAT-3’。
the sequencer in step 3 is specifically an ABI3730xl DNA Analyzer sequencer.
The beneficial effects of the invention are as follows: the method for identifying the cudweed plant variety by utilizing the ITS2 sequence provides a molecular identification basis for identifying cudweed medicinal materials, can accurately and rapidly detect cudweed and easily-mixed products thereof from a plurality of traditional Chinese medicinal materials, improves the detection accuracy, and is suitable for wide popularization and application.
Drawings
FIG. 1 is an amplified electrophoresis pattern of the Gnaphalium general primer ITS2F/3R in a method for identifying Gnaphalium plant species by using ITS2 sequences;
FIG. 2 is a graph of K2P genetic distances in a method of identifying a Gnaphalium plant variety using ITS2 sequences according to the present invention;
FIG. 3 is a Neighor Joining Tree chart of a method of the invention for identifying a Gnaphalium plant variety using the ITS2 sequence.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
The invention discloses a method for identifying a affine plant variety by utilizing an ITS2 sequence, which comprises the following steps:
step 1, taking a plurality of leaves of a plant sample to be identified, and extracting DNA of the leaves of the plant sample to be identified, as shown in a table 1, wherein the sources of the samples can be 8 species of cudweed (sequence numbers 1-8 in the table 1), 1 species of cudweed (sequence number 35 in the table 1) and 1 species of leontopodium styracifolium (sequence number 38 in the table 1) in different regions, and the samples are selected identified plant samples;
TABLE 1 plant sample Source list to be identified
Step 1.1, respectively taking a plurality of leaves of the plant sample to be identified, respectively placing the leaves into a centrifuge tube with the volume of 1.5mL, quick freezing the leaves in liquid nitrogen, then taking the leaves of the plant sample to be identified, placing the leaves into a plastic foam container containing the liquid nitrogen, and grinding the leaves into powder until the leaves are restored to room temperature;
step 1.2, preheating a CTAB solution with the concentration of 2% (m/V) to 65 ℃ in a water bath, taking 700ul of the CTAB solution, adding the 700ul of the CTAB solution into the plant sample leaves to be identified treated in the step 1, heating the plant sample leaves in the water bath with the temperature of 65 ℃ for 30min, taking the plant sample leaves out, and placing the plant sample leaves in a room temperature environment;
step 1.3, after the solution treated in the step 1.2 is restored to room temperature, 450ul of phenol chloroform is added, vortex shaking is carried out twice under the condition of 14000rpm for 10min each time, standing is carried out, 420ul of upper liquid is sucked, the upper liquid is placed into a centrifuge tube with the volume of 1.5mL, then chloroform with the same volume is added, vortex centrifugal shaking is carried out for 10min under the condition of 14000rpm, standing is carried out, layering is carried out, and supernatant is taken;
step 1.4, taking 300ul of the supernatant in the step 1.3, adding isopropanol precooled to the temperature of minus 20 ℃ in an equal volume, standing for not less than 10min at the temperature of minus 20 ℃, centrifuging for 10min at 14000rpm, standing, and discarding the supernatant to obtain a white precipitate at the bottom of the tube;
step 1.5, adding 500ul of ethanol with a volume fraction of 70% pre-cooled to-20deg.C into the white precipitate in step 1.4, suspending the precipitate, shaking at 14000rpm for 2min, standing at room temperature for 10min, discarding the supernatant, and adding 50ul of ddH 2 And O, centrifuging for 1min at 14000rpm, and preserving at the temperature of 4 ℃ to obtain the leaf DNA of the plant sample to be identified.
As shown in fig. 1: wherein the marker strips, the African mountain cudweed AH, the flower and fruit mountain cudweed H in the Han of Shanxi and the Tenebrio molitor TX are sequentially arranged from left to right; the DNA marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; as can be seen from FIG. 1, DNA amplification was successful and sequencing was possible.
Step 2, carrying out PCR amplification of the ITS2 nucleotide sequence on the DNA of the plant sample leaf to be identified in the step 1 to obtain a PCR product;
the PCR amplification reaction system is as follows: 2 XTaq PCR Mix 12.5ul, the common primers ITS2F and ITS3R are respectively 1ul with the concentration of 10umol/L, and 1ul of DNA obtained in the step 1 and 9.5ul of ddH2O are selected;
the reaction conditions for PCR amplification were: denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 45s,40 cycles; the temperature was 72℃for 2min.
The PCR amplification primers are as follows:
ITS2F：5’-ATCGATGCGATACTTGGTGTGAAT-3’，
ITS3R：5’-ATCGGACGCTTCTCCAGACTACAAT-3’。
step 3, performing bidirectional sequencing on the PCR product obtained in the step 2 by using an ABI3730xl DNA Analyzer sequencer to obtain a sequencing result, splicing and checking the sequencing result by using Seqman software, removing 5.8S and 28S sections at two ends by using a hidden Markov model annotation, and obtaining and storing the ITS2 spacer sequence of the plant sample leaf to be identified;
wherein the gene coding of the leaf ITS2 spacer sequence of the plant sample to be identified is shown as a sequence 1;
step 4, based on the leaf ITS2 spacer sequence of the plant sample to be identified obtained in the step 3, performing Blast sequence comparison in an NCBI database to obtain a homologous sequence and an outer edge seed sequence of the Gnaphalium plant, and obtaining rDNA ITS2 sequences of the homologous sequence and the outer edge seed sequence, wherein the rDNA ITS2 sequences are sources of the homologous sequence and the outer edge seed sequence as shown in a table 1;
and 5, aiming at the ITS2 sequences obtained in the step 3 and the step 4, performing data analysis by using MEGA7.0 software, calculating the K2P genetic distance, constructing a phylogenetic tree, and identifying the Gnaphalium plant variety.
As shown in fig. 2, the genetic distance between species was calculated using Kimura 2 parametric model; the genetic distance between the seeds is 0.0226-0.1468, the genetic distance between the seeds is 0-0.0100, and the minimum genetic distance between the seeds is larger than the maximum genetic distance between the seeds.
As shown in figure 3, for the constructed phylogenetic tree, it is obvious from the figure that the Gnaphalium affinis and the alien and miscible varieties thereof are respectively on different branches and can be obviously identified and separated.
The method for identifying the cudweed plant variety by utilizing the ITS2 sequence provides a molecular identification basis for identifying cudweed medicinal materials, can accurately and rapidly detect cudweed and easily-mixed products thereof from a plurality of traditional Chinese medicinal materials, improves the detection accuracy, and is suitable for wide popularization and application.
<110> Seiran medical college
<120> a method for identifying a Gnaphalium plant variety using ITS2 sequence
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 219
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
cgcatcgtgt cgccccctac aactcctcaa atggatgttt ggtgtggggg cggatattgg 60
tctcccgttt ctccgatatg gttggccaaa atacgagtcc cattcgatgg atgcacgact 120
agtggtggtt gctaaaacct tcgtcttcgg ttgtgcatct tcattcgtaa gggatgctta 180
aagaccccaa tgtgttgtct tctgatgacg cttcgaccg 219
Claims (5)
1. A method for identifying a cudweed plant variety using ITS2 sequence, comprising the steps of:
step 1, taking a plurality of leaves of a plant sample to be identified, and extracting DNA of the leaves of the plant sample to be identified;
step 2, carrying out PCR amplification of ITS2 nucleotide sequences on the DNA of the plant sample leaf to be identified in the step 1 to obtain a PCR product, wherein the PCR amplification primers are as follows:
ITS2F：5’-ATCGATGCGATACTTGGTGTGAAT-3’，
ITS3R：5’-ATCGGACGCTTCTCCAGACTACAAT-3’；
step 3, performing bidirectional sequencing on the PCR product obtained in the step 2 through a sequencer to obtain a sequencing result, splicing and checking the sequencing result through Seqman software, removing 5.8S and 28S sections at two ends through hidden Markov model annotation, and obtaining and storing the ITS2 spacer sequence of the plant sample leaf to be identified;
wherein the gene coding of the leaf ITS2 spacer sequence of the plant sample to be identified is shown as a sequence 1;
step 4, based on the leaf ITS2 spacer sequence of the plant sample to be identified obtained in the step 3, performing Blast sequence comparison in an NCBI database to obtain a homologous sequence and an outer edge seed sequence of the Gnaphalium plant, and obtaining rDNA ITS2 sequences of the homologous sequence and the outer edge seed sequence;
and 5, calculating K2P genetic distance according to the ITS2 sequences obtained in the step 3 and the step 4, constructing a phylogenetic tree, and identifying the Gnaphalium plant variety.
2. The method for identifying a cudweed plant variety using ITS2 sequence according to claim 1, wherein step 1 specifically comprises:
step 1.1, taking a plurality of plant sample leaves to be identified, respectively placing the plant sample leaves into a centrifuge tube with the volume of 1.5mL, placing the centrifuge tube into liquid nitrogen for quick freezing, then taking out the plant sample leaves to be identified, placing the plant sample leaves into a plastic foam container containing the liquid nitrogen, and grinding the plant sample leaves into powder until the plant sample leaves are restored to room temperature;
step 1.2, preheating CTAB solution with the concentration of 2% by mass/volume to 65 ℃ in a water bath, taking 700ul of the CTAB solution, adding the 700ul of the CTAB solution into the plant sample leaves to be identified treated in the step 1, heating the plant sample leaves in the water bath at 65 ℃ for 30min, taking the plant sample leaves out, and placing the plant sample leaves in a room temperature environment;
step 1.3, after the solution treated in the step 1.2 is restored to room temperature, 450ul of phenol chloroform is added, vortex shaking is carried out twice under the condition of 14000rpm for 10min each time, standing is carried out, 420ul of upper liquid is sucked, the upper liquid is placed into a centrifuge tube with the volume of 1.5mL, then chloroform with the same volume is added, vortex centrifugal shaking is carried out for 10min under the condition of 14000rpm, standing is carried out, layering is carried out, and supernatant is taken;
step 1.4, taking 300ul of the supernatant in the step 1.3, adding isopropanol precooled to the temperature of minus 20 ℃ in an equal volume, standing for not less than 10min at the temperature of minus 20 ℃, centrifuging for 10min at 14000rpm, standing, and discarding the supernatant to obtain a white precipitate at the bottom of the tube;
step 1.5, adding 500ul of ethanol which is precooled to-20 ℃ and has the volume fraction of 70% into the white precipitate in step 1.4, suspending the precipitate, oscillating for 2min at 14000rpm, standing for 10min at room temperature, discarding the supernatant, adding 50ul of ddH2O, centrifuging for 1min at 14000rpm, and preserving at the temperature of 4 ℃ to obtain the plant sample leaf DNA to be identified.
3. The method for identifying a variety of Gnaphalium plants by using ITS2 sequences according to claim 1, wherein the PCR amplification reaction system of the step 2 is as follows:
2 XTaq PCR Mix 12.5ul, and the common primers ITS2F and ITS3R each had a concentration of 10umol/L each of 1ul, and the DNA obtained in step 1 was 1ul and ddH2O 9.5ul.
4. A method for identifying a species of a affinis plant using ITS2 sequence according to claim 3, wherein the reaction conditions for PCR amplification in step 2 are:
denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 45s,40 cycles; the temperature was 72℃for 2min.
5. The method for identifying a variety of Gnaphalium plants using ITS2 sequences according to claim 1, wherein the sequencer in step 3 is specifically an ABI3730xl DNA Analyzer sequencer.
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| CN113160893B (en) * | 2021-06-09 | 2022-08-19 | 中国科学院昆明植物研究所 | Mining plant ITSs sequence from second generation sequencing data and using the same for identifying variety families |
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