阅读说明：本技术 一种果上叶dna的提取方法 (Method for extracting DNA from fruit upper leaves ) 是由 杨建波 杨佳红 张旭 罗云 杨艳 张道琴 于 2021-10-20 设计创作，主要内容包括：本发明属于植物DNA提取的技术领域,具体涉及一种果上叶DNA的提取方法,包括步骤：(1)将果上叶置于聚丙烯酰胺溶液中搅拌10-20min,取出冲洗,表面喷洒非离子表面活性剂溶液,再经液氮速冻后,研磨成粉末；(2)向粉末中加DNA提取溶液进行水浴提取；(3)加抽提溶液混匀,离心,取上清液A；(4)向上清液A中加氯仿/异戊醇混合物混匀,离心,取上清液B；(5)向上清液B中加无水乙醇混匀,静置、离心,析出沉淀、水洗；本发明方法具有DNA提取率高、杂质少的特点,符合后续DNA分子学鉴定的要求。(The invention belongs to the technical field of plant DNA extraction, and particularly relates to a method for extracting DNA of fruit leaves, which comprises the following steps: (1) placing the upper leaves of the fruits in a polyacrylamide solution, stirring for 10-20min, taking out and washing, spraying a nonionic surfactant solution on the surface, quickly freezing by liquid nitrogen, and grinding into powder; (2) adding DNA extraction solution into the powder for water bath extraction; (3) adding the extraction solution, mixing uniformly, centrifuging, and taking supernatant A; (4) adding a chloroform/isoamyl alcohol mixture into the supernatant A, uniformly mixing, centrifuging, and taking a supernatant B; (5) adding absolute ethyl alcohol into the supernatant B, uniformly mixing, standing, centrifuging, separating out a precipitate, and washing with water; the method has the characteristics of high DNA extraction rate and less impurities, and meets the requirement of subsequent DNA molecular identification.)

1. A method for extracting DNA from fruit leaves is characterized by comprising the following steps:

(1) according to the following steps of 1: (1.3-1.8) placing the upper leaves of the fruits in a polyacrylamide solution according to the mass ratio of the feed liquid, stirring for 10-20min, taking out and washing for 1-2 times, spraying a nonionic surfactant solution with the mass of 0.1-0.5% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding a DNA extraction solution preheated to the water bath temperature, shaking uniformly, carrying out water bath for 30-60min, and reversing for 3-5 times during the water bath;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.8-1 time volume of precooled absolute ethyl alcohol, fully reversing and uniformly mixing, standing at-20 ℃ for 10-20min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing the DNA precipitate 1-2 times.

2. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the polyacrylamide solution has a volume concentration of 10-30%.

3. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the volume concentration of the non-ionic surfactant solution is 40-50%.

4. The method for extracting DNA from fruit leaves according to claim 1, wherein the non-ionic surfactant is any one of Tween 20, Tween 40, Tween 60, Tween 80, span-20 and span-80.

5. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the DNA extraction solution is 2-3% CTAB, 0.08% -0.1% (V/V) β -mercaptoethanol, 0.01% -0.04% anionic surfactant, 1.1-1.3 mol/LNaCl.

6. The method for extracting DNA from fruit leaves as claimed in claim 5, wherein the anionic surfactant is sodium dodecylbenzenesulfonate.

7. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the extraction solution is a mixture of phenol, chloroform and isoamyl alcohol at a volume ratio of 25:24: 1.

8. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the temperature of the water bath is 55-60 ℃.

9. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the chloroform/isoamyl alcohol mixture is a mixture of chloroform to isoamyl alcohol at a volume ratio of 24: 1.

10. The method for extracting DNA from fruit leaves as claimed in claim 1, wherein the temperature of the precooled absolute ethyl alcohol is 5-10 ℃.

Technical Field

The invention belongs to the technical field of plant DNA extraction, and particularly relates to a method for extracting DNA from fruit leaves.

Background

The fruit upper leaves are the variety accepted in the quality standards of traditional Chinese medicinal materials and national medicinal materials in Guizhou province (2003 edition), are derived from dry rhizome and pseudobulb of Yunnan stone peach Pholidota yunnanensis Rolfe in the family of orchid, have the effects of nourishing yin, clearing away the lung-heat, eliminating phlegm, relieving cough, promoting qi circulation and relieving pain, and are used for treating cough and hemoptysis due to pulmonary tuberculosis, chronic tracheitis, chronic pharyngitis, hernia pain, sore and ulcer swelling and pain and the like.

At present, from the view of collected wild fruit upper leaf resources, the wild fruit upper leaf resources have complex diversity, and the wild fruit upper leaf resources are difficult to accurately identify only from morphological identification, so that the identification accuracy can be improved by adopting biological identification, and the quality of the obtained DNA is poor and the subsequent identification is difficult to meet by adopting the existing DNA extraction method.

Disclosure of Invention

The invention provides a method for extracting DNA from fruit leaves aiming at the defects of the prior art.

The method is realized by the following technical scheme:

a method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: (1.3-1.8) placing the upper leaves of the fruits in a polyacrylamide solution according to the mass ratio of the feed liquid, stirring for 10-20min, taking out and washing for 1-2 times, spraying a nonionic surfactant solution with the mass of 0.1-0.5% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding a DNA extraction solution preheated to the water bath temperature, shaking uniformly, carrying out water bath for 30-60min, and reversing for 3-5 times during the water bath;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.8-1 time volume of precooled absolute ethyl alcohol, fully reversing and uniformly mixing, standing at-20 ℃ for 10-20min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing the DNA precipitate 1-2 times.

The volume concentration of the polyacrylamide solution is 10-30%.

The volume concentration of the non-ionic surfactant solution is 40-50%.

The non-ionic surfactant is any one of Tween 20, Tween 40, Tween 60, Tween 80, span-20 and span-80.

The DNA extraction solution is 2-3% CTAB, 0.08% -0.1% (V/V) beta-mercaptoethanol, 0.01% -0.04% anionic surfactant and 1.1-1.3 mol/LNaCl.

The anionic surfactant is sodium dodecyl benzene sulfonate.

The extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1.

The temperature of the water bath is 55-60 ℃.

The chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1.

The temperature of the precooled absolute ethyl alcohol is 5-10 ℃.

Has the advantages that:

the method has the characteristics of high DNA extraction rate and less impurities, and meets the requirement of subsequent DNA molecular identification.

According to the characteristics of chemical component content of the leaves on the fruits, the invention researches out the extraction method suitable for the DNA of the leaf groups on the fruits, reduces the interference of secondary metabolites such as polysaccharide, polyphenol and the like on the extraction step of the complete DNA, simultaneously reduces the extraction temperature, avoids the damage of the temperature on the DNA, improves the integrity rate of the DNA, and is specifically embodied in that:

1) the invention utilizes polyacrylamide to remove surface impurities.

2) The invention improves the pore structure of the upper leaves of the fruits by using a non-ionic surfactant and a liquid nitrogen quick cooling method, so that the DNA is easy to dissolve out.

3) According to the invention, by optimizing the formula of the DNA extraction solution, particularly adding the anionic surfactant, the hydrolysis and combination conditions of substances such as protein, polysaccharide, fiber and the like are greatly improved, the solubilization effect of components such as DNA and the like is improved, the extraction temperature is reduced, and the decomposition and damage of DNA at higher temperature are avoided.

4) In the presence of high salt, CTAB is combined with protein, polysaccharide and other substances, but not combined with DNA, thereby further ensuring the integrity of DNA.

5) The method uses the absolute ethyl alcohol, avoids the phenomenon that the isopropanol precipitates DNA and is simultaneously coprecipitated with salts, saccharides and the like, and can be washed by water because the ethyl alcohol is easy to volatilize, thereby avoiding the washing of the ethyl alcohol and reducing the use of chemical reagents.

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

A method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: 1.3, placing the upper leaves of the fruits in a polyacrylamide solution with the volume concentration of 10%, stirring for 10min, taking out and washing for 1 time, spraying a Tween 20 solution with the mass of 0.1% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder; the volume concentration of the Tween 20 solution is 40%;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding a DNA extraction solution with the temperature of 55 ℃, shaking uniformly, carrying out water bath for 30min at the temperature of 55 ℃, and reversing for 3 times during the water bath; the DNA extraction solution is 2% CTAB, 0.08% (V/V) beta-mercaptoethanol, 0.01% sodium dodecyl benzene sulfonate and 1.1 mol/LNaCl;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1; the extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1; the chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.8 time volume of 5 ℃ absolute ethanol, fully reversing and uniformly mixing, standing at-20 ℃ for 10min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing the DNA precipitate 1 time.

Example 2

A method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: 1.8, placing the upper leaves of the fruits in a polyacrylamide solution with the volume concentration of 30%, stirring for 20min, taking out and washing for 2 times, spraying a Tween 40 solution with the mass of 0.5% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder; the volume concentration of the Tween 40 solution is 50%;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding the DNA extraction solution with the temperature of 60 ℃, shaking uniformly, carrying out water bath for 60min at the temperature of 60 ℃, and reversing for 5 times during the water bath; the DNA extraction solution is 3% CTAB, 0.1% (V/V) beta-mercaptoethanol, 0.04% sodium dodecyl benzene sulfonate and 1.3 mol/LNaCl;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1; the extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1; the chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1;

(5) transferring the supernatant B into a new centrifuge tube, adding 1 time volume of 10 ℃ absolute ethyl alcohol, fully reversing and uniformly mixing, standing at-20 ℃ for 20min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing DNA precipitation 2 times.

Example 3

A method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: 1.4, placing the upper leaves of the fruits in a polyacrylamide solution with the volume concentration of 15%, stirring for 18min, taking out and washing for 2 times, spraying a span-20 solution with the mass of 0.4% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder; the volume concentration of the span-20 solution is 48 percent;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding a DNA extraction solution with the temperature of 58 ℃, shaking uniformly, carrying out water bath for 50min at the temperature of 58 ℃, and reversing 4 times during the water bath; the DNA extraction solution is 2.5 percent of CTAB, 0.08 percent (V/V) of beta-mercaptoethanol, 0.04 percent of sodium dodecyl benzene sulfonate and 1.2 mol/LNaCl;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1; the extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1; the chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.9 times of anhydrous ethanol with the volume of 8 ℃, fully reversing and uniformly mixing, standing at-20 ℃ for 15min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing the DNA precipitate 1 time.

Example 4

A method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: 1.5, placing the upper leaves of the fruits in a polyacrylamide solution with the volume concentration of 20%, stirring for 15min, taking out and washing for 1 time, spraying a span-80 solution with the mass of 0.3% of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder; the volume concentration of the span-80 solution is 45 percent;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding DNA extraction solution with the temperature of 57 ℃, shaking uniformly, carrying out water bath for 45min at the temperature of 57 ℃, and reversing for 4 times during the water bath; the DNA extraction solution is 2.5 percent of CTAB, 0.09 percent (V/V) of beta-mercaptoethanol, 0.02 percent of sodium dodecyl benzene sulfonate and 1.2 mol/LNaCl;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1; the extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1; the chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.9 times of anhydrous ethanol with the volume of 8 ℃, fully reversing and uniformly mixing, standing at-20 ℃ for 15min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing DNA precipitation 2 times.

Example 5

A method for extracting DNA from fruit leaves comprises the following steps:

(1) according to the following steps of 1: 1.6, placing the upper leaves of the fruits in 25 volume percent polyacrylamide solution, stirring for 13min, taking out and washing for 2 times, spraying Tween 80 solution with the mass of 0.2 percent of the upper leaves of the fruits on the surface, quickly freezing by liquid nitrogen, and grinding into powder; the volume concentration of the Tween 80 solution is 45%;

(2) pouring the powder of the upper leaves of the fruits into a cold EP tube, adding DNA extraction solution with the temperature of 59 ℃, shaking uniformly, carrying out water bath for 32min at the temperature of 59 ℃, and reversing for 3 times during the water bath; the DNA extraction solution is 2% CTAB, 0.1% (V/V) beta-mercaptoethanol, 0.01% sodium dodecyl benzene sulfonate and 1.1 mol/LNaCl;

(3) taking out the EP tube, cooling to room temperature, adding the extraction solution, fully reversing, uniformly mixing, centrifuging at 12000 Xg for 5min, and taking supernatant A; the volume ratio of the extraction solution to the DNA extraction solution is 1: 1; the extraction solution is a mixture of phenol, chloroform and isoamylol in a volume ratio of 25:24: 1;

(4) transferring the supernatant A into a new EP tube, adding a chloroform/isoamylol mixture, fully reversing and uniformly mixing, centrifuging at 12000 Xg for 5min, and taking a supernatant B; the volume ratio of the chloroform/isoamyl alcohol mixture to the supernatant A is 1: 1; the chloroform/isoamyl alcohol mixture is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24: 1;

(5) taking the supernatant B, transferring the supernatant B into a new centrifuge tube, adding 0.8 times volume of 7 ℃ absolute ethyl alcohol, fully reversing and uniformly mixing, standing at-20 ℃ for 12min, and centrifuging at 12000 Xg for 10min to fully separate out DNA to obtain DNA precipitate;

(6) washing the DNA precipitate 1 time.

Experimental example 1

After the DNA precipitates obtained in the examples 1 and 3 are volatilized to be dry, dissolving the DNA precipitates by using 100 mu LTE, and checking the extraction condition of the DNA by using gel electrophoresis, wherein the bands detected by the gel electrophoresis are bright, clear and have no band drop; and simultaneously, an ultraviolet absorption method is utilized for detection and display: the DNA precipitates obtained in example 1 and example 3 each contained no impurities such as proteins and polysaccharides; generally, 0.04g of powder of fruit upper leaves is treated with 600 μ L of DNA extraction solution, after 1.00g of fruit upper leaves are extracted, about 32.10 μ g of DNA is obtained in example 1, about 31.06 μ g of DNA is obtained in example 3, other example groups are tested by the same method, the situation is the same, and the DNA extraction amount of 1.00g of fruit upper leaves is 31.00-32.10 μ g, and the DNA extraction amount of each example group has no significant difference.

Experimental example 2

The following fruit upper leaf samples were identified using the method of example 2; the sample conditions were as follows:

sample 1: the rhizomes are stoloniferous, thick and strong, 2-6 mm thick, and residual fibers are generated after the rhizomes are rotten by the goblet-shaped membranous sheaths or the sheaths. Pseudobulb is on the root stem, the egg-shaped cone or the narrow egg shape, 1 leaf is grown on the top, and the stem is gray brown after being dried. Leathery, oblong. The scape yellow and green color band is purplish red stripe, which is extracted from the base of the false bulb and is upright. Pseudobulb grows or is close to the root stem, and is nearly cylindrical, 1 leaf is grown at the top, and the fiber remains after the base part is rotten by the sheath. The leaf leathery, long and narrow, round, nearly sessile, rib bulging in the back.

Sample 2: the rootstock crawls, and 1 stem is sent out every 3-6 internodes. The stem is yellow or yellow brown, usually drooping, and multi-branched. The pseudobulb is golden yellow, is slightly flat and spindle-shaped, has 1 internode and is provided with 1 leaf by top growth. The leatheroid, oblong or oblong, is needle-shaped.

Sample 3: the pseudobulb is dense, oval, slightly flat, oval and long-round in leaf shape, is leathery, slightly fleshy, and the scape is 10-20 cm long; the inflorescence handle is nearly cylindrical, has no obvious wings and no immature bracts; the raceme usually has more than 10 flowers, is light orange red and is slightly separated, and the flowering period is 10-11 months.

Sample 4: the rhizomes are creeping, the pseudobulbs are dense and approximately oblong, and 1 leaf is arranged at the top end or the end close to the top end.

Sample 5: the rootstock is slender and creeping. The pseudobulb is narrow in shape to be nearly cylindrical, and 2 leaves are grown at the top end. The leaves are round or long and round and are coated with needle-shaped paper.

Sample 6: the rootstock is slender and creeping. The pseudobulb is narrow in shape to be nearly cylindrical, and 2 leaves are grown at the top end. The leaves are round or long and round and are coated with needle-shaped paper. The scape is emitted from the top end of the grown pseudobulb and is 5-10 cm long; the raceme usually has 1-2 flowers, the flower is light yellow, and only the lipsticks have red marks; the petals are in a filiform or a narrow line shape and are approximately equal to sepals in length; the labial lobe is oval, 3 fissures.

Sample 7: the rhizomes are creeping and often branched, the pseudobulb is slender and fusiform, 1 leaf grows from the top end, and the size is small. There are many fibrous roots.

Sample 8: the rootstock is slender and creeping. The pseudobulb is narrow in shape to be nearly cylindrical, and 2 leaves are grown at the top end. The leaves are round or long and round and are coated with needle-shaped paper. The scape is emitted from the top end of the grown pseudobulb and is 5-10 cm long; the raceme usually has 1-2 flowers, the flower is light yellow, and only the lipsticks have red marks; the petals are in a filiform or a narrow line shape and are approximately equal to sepals in length; the labial lobe is oval, 3 fissures.

Sample 9: pseudobulb is dense, nearly narrow-pointed or oval, inverted needle-shaped or inverted needle-shaped with narrow-pointed shape for leaves, and hard paper, and scape is 11-25 cm long;

sample 10: the rootstock is creeping, frequently branched, pseudobulb oval, leaf scaly, thick leathery, scape is born on the top of young pseudobulb, and young leaves are sent out from the rootstock near the base of old pseudobulb.

Sample 11, root tuber creeping, branching, dense scale sheath, node thinning and rooting; pseudobulbar narrow oval to oval oblong with 2 leaves growing at the tip. Leaf line shape or linear shape is coated with needle shape and paper. The scape is grown on the top of young false bulb, and the base and young leaves are wrapped by the sheath when the scape is sent out.

Sample 12: the rootstock is creeping, sheathed when young, and the old fiber remained on the node by the sheath, and the root comes out of the rootstock node with the pseudobulb. The pseudobulb is oval, the hard straight fiber remained after the base part is rotten by the sheath, the yellow fiber is dry and has longitudinal edges, thick and leathery leaves, long and narrow circles, and the petioles are folded in half.

The DNAs extracted from the above samples were subjected to ITS1-5.8SrDNA-ITS2 sequence PCR reaction, and ITS1-5.8SrRNA-ITS4 sequences of fungi were amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as primers according to the reaction system of Table 1 under the following reaction conditions.

Then, the amplification product was electrophoretically amplified using 1% (w/V) agarose gel at 100V for 30min to check whether the amplification was successful.

TABLE 1 PCR reaction System

Sequence analysis: and (3) carrying out PCR electrophoresis detection, then sending the obtained PCR electrophoresis detection to Invitrogen (Shanghai, China) or the Ongke biotechnology limited company (Beijing, China) for sequencing, submitting the sequenced sequence to a NCBI Genebank database for BLAST comparison, selecting a representative strain with the largest similarity according to the comparison result, and carrying out cluster analysis by using a Neighbor-join (NJ) method by utilizing MEGA5.10 software.

The results show that:

sample 1: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the ITS1-5.8SrDNA-ITS2 sequence and Bulbophyllum andersonii isolate SMSDLITS (Accession No. JN619417.1) is the largest and reaches 98%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 1 as the comb cap convolvulus blue B.andersonii according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 2: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity with Dendrobium album isocyanate CT2(Accession No.: MK483264.1) is the largest and reaches 97%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 2 as D.albopurpureum according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 3: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the Liparis guangxiensis voucher L.Li 153(access No. KF589875.1) similarity is the largest and reaches 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 3 as the L.guangxiensis of the euryale ferox according to the Blast comparison result, the phylogenetic tree analysis result and the morphological characteristics of the sample.

The plants in sample 4 and sample 5 were completely the same in morphology, and were the same plants: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the sequence and L.balansae voucher L.Li 152(access No. KF589874.1) and L.latilabris voucher Z.J.Liu 4770(access No. KJ459291.1) is maximum and reaches 98 percent. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. According to the Blast comparison result and the phylogenetic tree analysis result, the sample 4 and the sample 5 cannot be clustered with a certain sequence independently, and the sample 4 and the sample 5 are initially identified as the allium sativum Liparis sp.

Sample 6: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and then the obtained sequence is found to have the maximum similarity with Coelogyne fimbriata vorher L.Li 11(IBSC) (access No. KR857330.1) and C.fimbriata var.leungian vor BG260(access No. KY966507.1), and both the similarity reach 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And preliminarily identifying the sample 6 as the fritillaria fimbriata C.fimbriata according to the Blast comparison result and the phylogenetic tree analysis result and referring to morphological characteristics of the sample.

Sample 7: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the ITS1-5.8SrDNA-ITS2 sequence and Bulbophyllum sheweiliens voucher Jian-Wu _ Li1636(Accession No. MK164507.1) is the maximum and reaches 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 7 as the bulbophyllum umbellate B.shweiliense W.W.Sm according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 8: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and then the similarity of the sequence and Coelogyne fimbriata vorcher KFBG712(Accession No. KY966506.1) and C.fimbriata vorher L.Li 11(IBSC) (Accession No. KR857330.1) is found to be maximum and reaches 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 8 as the fritillaria fringensis C.fimbriata according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 9: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBIGenBank database data, and the similarity of the sequence and L.balansae voucher L.Li 152(Accession No. KF589874.1) is the maximum and reaches 97%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 9 as the plant of the genus allium capilalum Liparis sp according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 10: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the ITS1-5.8SrDNA-ITS2 sequence and Pholidota mieriorum voucher KFBG225(access No. KY966652.1) is found to be the maximum and reaches 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 10 as the P.missionariorum according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 11: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the sequence and P.cantonensis voucher KFBG658A (Accession No. KY966649.1) is found to be maximum and reaches 96%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. And identifying the sample 11 as P.cantonensis according to the Blast comparison result, the phylogenetic tree analysis result and morphological characteristics of the sample.

Sample 12: the ITS1-5.8SrDNA-ITS2 sequence obtained by amplification is subjected to Blast comparison on NCBI GenBank database data, and the similarity of the sequence and Bulbophyllum tianguii isolate TGSDLITS (Accession No. JN619415.1) is found to be the maximum and reaches 99%. Meanwhile, a phylogenetic tree is constructed by adopting MEGA5.10 software and using N-J, and the self-development value is set to be 1000, so that the phylogenetic tree is obtained. Sample 12 was identified as celadon blue b.tianguii based on Blast alignment and phylogenetic tree analysis, as well as its morphological characteristics.

In the above identification, all plants were identified as species except that sample 4, sample 5 and sample 9 identified only genus, and the remaining 9 materials were classified into 8 different species.