CN113151418A - Method for identifying and identifying kokstroemia indica based on morphology and molecular identification - Google Patents
Method for identifying and identifying kokstroemia indica based on morphology and molecular identification Download PDFInfo
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- CN113151418A CN113151418A CN202110321633.2A CN202110321633A CN113151418A CN 113151418 A CN113151418 A CN 113151418A CN 202110321633 A CN202110321633 A CN 202110321633A CN 113151418 A CN113151418 A CN 113151418A
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- C12Q1/6858—Allele-specific amplification
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- G06V10/74—Image or video pattern matching; Proximity measures in feature spaces
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Abstract
The invention discloses a method for identifying and identifying hevea brasiliensis based on morphology and molecular identification, which comprises the steps of selecting complete plant tissues of the hevea brasiliensis to be detected; selecting the rubber grass to be detected, and carrying out morphological observation and primary screening to obtain the rubber grass which is screened for the first time; performing molecular identification on the screened turfgrass, and performing statistics and analysis on obtained data to output a result; and identifying the rubber grass to be detected through the result identification. The method can combine morphological observation and molecular level joint identification, can eliminate hybrid interference of the dandelion and the kochia scoparia, is suitable for collecting the plant materials of the wild kochia scoparia and can also be used for preserving pure germplasm of the kochia scoparia.
Description
Technical Field
The invention belongs to the technical field of plant tissue identification, and particularly relates to a method for identifying hevea brasiliensis based on morphological and molecular identification.
Background
Kokuba, wherein mevalonic acid (MVA) is used in the pathway for regulation of natural rubber biosynthesis, is diploid (2n ═ 16), has a relatively simple genome, is 1.29Gb in length, and comprises 46,731 predicted protein-encoding genes, and kokuba roots contain high molecular weight rubber molecules, with properties comparable to those of the rubber latex of hevea brasiliensis, and can be used as a substitute rubber crop, with high rubber content of kokuba roots, accounting for 3% to 28% of their total dry weight [17], which are also new sources of inulin (linear β - (2-1) -linked fructan), another abundant metabolite of dandelion, stored in parenchymal root cell vacuoles near the phloem, adjacent to extraplasmically isolated latex worms, an important material for the food industry and bioethanol production. In addition, the rubber grass can be widely grown in cold and temperate regions in a relatively short life cycle and easily harvested and genetically transformed, and thus can be used as an ideal model plant for the gene function of the wheat of the invention. A biosynthetic material of natural rubber.
However, because the characteristics of the kokspur plants are easily confused with other dandelions, the common dandelion leaves are pinnate, the edges are saw-toothed, the base is wedge-shaped, and the kokspur leaves are wedge-shaped, and the leaf edges are almost smooth and round. A large number of latex cells and rubber particles were detected in many rubber grass tissues and the dried roots of the rubber grass contained a large amount of natural rubber, the latex cells and rubber particles were only detected in the veins, stems and roots of the rubber grass, with an average molecular weight of 2180kDa in the root latex, which is very similar to that obtained from dandelion. Therefore, it is difficult to distinguish between Hemsleya chinensis and Taraxacum mongolicum by simply washing the molecular weight. There is therefore a need for a method for identifying kochia based on morphological and molecular identification.
Disclosure of Invention
The invention provides a method for eliminating hybrid interference of dandelion and hevea brasiliensis, and identifying hevea brasiliensis based on morphological and molecular identification.
The invention comprises the following steps:
a, selecting complete plant tissues of the rubber grass to be detected;
b, selecting the rubber grass to be detected, and carrying out morphological observation and primary screening to obtain the rubber grass which is screened for the first time;
c, performing molecular identification on the screened rubber grass, and performing statistics and analysis on obtained data to output a result;
d, identifying the rubber grass to be detected through the result identification;
further, the method for molecular identification comprises
a, extracting chloroplast from the hevea brasiliensis to be detected by a chromatography method;
b, extracting the total DNA of the hevea brasiliensis chloroplast, and detecting by using the species specificity marker C5 gene of the hevea brasiliensis chloroplast for PCR;
c, detecting through 1.2% agarose gel electrophoresis, which shows that the right bands (659bp) of the rubberella and the common dandelion,
d, performing single enzyme digestion on the PCR product by using TaqI enzyme, generating polymorphism phenomena of a plurality of indefinite fragments in a product of the rubber grass (C5), and generating polymorphism bands after enzyme digestion, thereby determining the rubber grass as the PCR product.
Further, the method for morphological observation comprises
A, obtaining a characteristic image of standard hevea brasiliensis, and selecting a clear graph with obvious characteristics;
b, recording and storing the image data of the standard rubber grass as standard image data for comparing the images of the rubber grass to be detected and storing the standard image data in a plant characteristic database;
c, when the rubber grass needs to be identified and identified, calculating and comparing data corresponding to the rubber grass characteristic graph by using a rubber grass model in a plant characteristic database or measuring the data by using the same method in a real-time field;
further, the reaction system is:
DNA 2μL
TaKaRa 1×PCRMix7μL
Primers 1μL
ddH2O 10μL
Totalvolume 20.0μL
the reaction conditions were set as follows: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 deg.C, 30sec, annealing at 54 deg.C, 30sec, extension at 72 deg.C, 1min, and circulation for 30 times; 72 ℃ for 10 min; storing at 4 ℃.
Further, PCR reaction was carried out using a caerulea Chloroplast (Chloroplast) gene specific primer C5(Interspacebetweentrn N-GUUandrps15) as a detection primer, and the sequence of the primer was: annotation C5 (Interspaacebetweentrn N-GUUandrps15)
Forwardprimer:5′-TCAAAGGATCTATGCGCAATCA-3′
Reverseprimer:5′-TCGAGAATTGAAGACCCCTAGT-3′
The total Length of the C5 gene is 462bp, time is 54 ℃,
Enzyme:TaqI,TKS:Polymorphic,TO:
the invention has the beneficial effects that:
the method can combine morphological observation and molecular level joint identification, can eliminate hybrid interference of the dandelion and the kochia scoparia, is suitable for collecting the plant materials of the wild kochia scoparia and can also be used for preserving pure germplasm of the kochia scoparia.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
fig. 2 is a picture showing chloroplast extraction.
FIG. 2A. Taraxacum chloroplast; FIG. 2B. Hedychium notatum chloroplast
FIG. 3 is a schematic diagram of chloroplast-specific C5 primer validation.
Wherein FIG. 3A. the PCR results of the C5 gene are shown schematically; FIG. 3B shows the single-restriction enzyme C5 gene polymorphism verification
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
As shown in fig. 1, the present embodiment includes the following steps:
a, selecting complete plant tissues of the rubber grass to be detected, and respectively collecting leaves, buds, rhizomes and seeds of the plants;
b, selecting the rubber grass to be detected, and carrying out morphological observation and primary screening to obtain the rubber grass which is screened for the first time;
common dandelion and taraxacum are of the taraxacum genus and initially appear to be of the same species. The leaves of Hemsleya amabilis and Taraxacum officinale are in the shape of roses. The leaves of regular dandelion are pinnate but the leaves of hevea brasiliensis are more wedge-shaped. The edges of the common dandelion leaves are ragged and the base is wedge-shaped. The blade tip is sharp and the base of the main vein is reddish brown in the form of a lateral parallel vein. The shape of the rubber grass blade is also wedge-shaped, the edge of the blade is almost smooth and round, the blade base is also wedge-shaped, and the blade tip is a half circle. The base of the main vein is white or red, also in lateral form parallel to the vein.
The dandelion flowers are capitate in shape, but there are significant differences in morphology between the common dandelion and the outer pieces of the hevea brasiliensis bud. The outer sheet of the common dandelion bud extends outwards, while the outer sheet of the rubber grass bud forms an angle inwards. The seeds and spindle morphology of dandelion under optical microscope were observed to be of lean fruit. Common dandelion and kombucha seeds are dark brown and dark green, respectively. The seed surface is morphologically arranged in the profile of ridges and furrows, and barbs appear in the latter half of the ridges. The morphological characteristics of the seeds facilitate rapid penetration into the soil after landing. Because the rubber grass has self incompatibility, the surface contour of the fertilized seed of the rubber grass is clear, and a single seed is large and full; the common dandelion root fracture has no colloid wiredrawing, and has continuous white colloid line graph of the rubber grass dry root fracture.
Identifying the hevea brasiliensis by a morphological observation method:
the common dandelion and the rubber grass flower bud have obvious difference. The rubber grass sheet is in an inverted needle shape, the front end of the rubber grass sheet is blunt, the edge of the rubber grass sheet is smooth and curved, and the flower buds of the rubber grass sheet and the outer layer of the panel total wrapping sheet are curved inwards to form a certain angle. Kokspur was self-incompatible, its seeds were spindle-shaped thin fruits and showed vertical satiation.
1. Comparing wild hevea brasiliensis with dandelion, observing the whole plant vertically to be round, wherein the dandelion leaves have jagged gaps, the petioles are pink, and the whole leaf area is large;
2. the disc-shaped area of the rubber grass is small, the blades are not provided with saw-toothed gaps, the blades scatter the surrounding blades by taking stems as centers and are dark green, the blades are oval and thicker than common dandelion blades, the veins are white, no obvious gaps are arranged at the edges of the blades, and the whole blades are oval;
3. the flower buds of the Hedychium spicatum are smaller, and the sepals at the outermost layer of the calyx are internally buckled to form a certain angle.
4. The sepals on the bottom layer of the rubber grass flower buds are buckled inwards to form a certain angle;
5. comparing seeds, observing the shapes and colors of the seeds of the two plants, wherein the seeds of the common dandelion are brown, have longer barbs and have clear seed textures;
the seeds of the rubbergrass are dark green, the seeds are slender, the barbs are shorter, and the grains of the seeds are shallow; the pollinated seeds are obviously full, the seeds are large, the non-pollinated seeds are shrunken, and the seeds are small and light;
c, performing molecular identification on the screened rubber grass, and performing statistics and analysis on obtained data to output a result;
molecular level identification of Hevea brasiliensis (chloroplast)
(1) Using the obtained chloroplast DNA as a template and using the melissa officinalis chloroplast
(Chloroplast) gene specific primer C5 (Interspacebedweintrn-GUUandrps 15) as a detection primer was used for PCR reaction, and the sequence of the primer was:
Annotation:C5(InterspacebetweentrnN-GUUandrps15)
Forwardprimer:5′-TCAAAGGATCTATGCGCAATCA-3′
Reverseprimer:5′-TCGAGAATTGAAGACCCCTAGT-3′
the total Length of the C5 gene is 462bp, time is 54 ℃, Enzyme is TaqI, rubber grass is Polymorphic, common dandelion:
Fixed；
reaction system:
the reaction conditions were set as follows: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 deg.C, 30sec, annealing at 54 deg.C, 30sec, extension at 72 deg.C, 1min, and circulation for 30 times; 72 ℃ for 10 min; storing at 4 ℃.
(2) After the PCR reaction is finished, taking 5-8 mu L of PCR product reaction solution, detecting by agarose gel electrophoresis (agarose gel electrophoresis), confirming that the size of a PCR amplification product band is correct, and recording the size of the amplification product for later use; if the PCR amplification product is to be used in subsequent experiments, please store at-20 ℃. Molecular genetic identification can fully prove the purity of the hevea brasiliensis strain. In the invention, the Procoll gradient centrifugation method is used for extracting the chloroplast of the hevea brasiliensis leaves (figure 2), and the dandelion (common dandelion) and the chloroplast of the hevea brasiliensis are better extracted in quality by an optical microscope.
D, identifying the rubber grass to be detected through the result identification;
extracting chloroplast by using the chromatography, extracting total DNA of the melissa officinalis chloroplast by using a polysaccharide polyphenol plant DNA kit, detecting by using a species-specific marker C5 gene of the melissa officinalis chloroplast to perform PCR (figure 2A), detecting by using 1.2% agarose gel electrophoresis (agarose electrophoresis), displaying a correct band (659bp) of the melissa and common dandelion, performing single enzyme digestion on a PCR product by using TaqI enzyme, generating polymorphism of a plurality of indefinite fragments in a melissa (C5) product (figure 2B), and generating a polymorphism band after enzyme digestion, thereby determining the melissa. The method is used as a qRTPCR internal reference gene when the function of the hevea brasiliensis gene is verified, can also be used as an internal reference gene of semi-quantitative RT-PCR as a reference, and can also be used under the conditions of verifying whether hybridization exists between the hevea brasiliensis and common dandelion and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A method for identifying the kochia scoparia based on morphological and molecular identification is characterized by comprising the following steps:
a, selecting complete plant tissues of the rubber grass to be detected;
b, selecting the rubber grass to be detected, and carrying out morphological observation and primary screening to obtain the rubber grass which is screened for the first time;
c, performing molecular identification on the screened rubber grass, and performing statistics and analysis on obtained data to output a result;
and D, identifying the rubber grass to be detected through the result identification.
2. The method for identifying kokstroemia indica based on morphological and molecular identification as claimed in claim 1, wherein said molecular identification method comprises
a, extracting chloroplast from the hevea brasiliensis to be detected by a chromatography method;
b, extracting the total DNA of the hevea brasiliensis chloroplast, and detecting by using the species specificity marker C5 gene of the hevea brasiliensis chloroplast for PCR;
c, detecting through 1.2% agarose gel electrophoresis, which shows that the right bands (659bp) of the rubberella and the common dandelion,
d, performing single enzyme digestion on the PCR product by using TaqI enzyme, generating polymorphism phenomena of a plurality of indefinite fragments in a product of the rubber grass (C5), and generating polymorphism bands after enzyme digestion, thereby determining the rubber grass as the PCR product.
3. The method for identifying kokstroemia indica based on morphological and molecular characterization as claimed in claim 1, wherein said morphological observation method comprises
A, obtaining a characteristic image of standard hevea brasiliensis, and selecting a clear graph with obvious characteristics;
b, recording and storing the image data of the standard rubber grass as standard image data for comparing the images of the rubber grass to be detected and storing the standard image data in a plant characteristic database;
and C, when the rubber grass needs to be identified and identified, calculating and comparing the data corresponding to the rubber grass characteristic graph by using a rubber grass model in a plant characteristic database or measuring the data by using the same method in a real-time field.
4. The method for identifying kochia based on morphological and molecular identification as claimed in claim 2, wherein the reaction system is:
DNA 2μL
TaKaRa 1×PCRMix7μL
Primers 1μL
ddH2O 10μL
Totalvolume 20.0μL
the reaction conditions were set as follows: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 deg.C, 30sec, annealing at 54 deg.C, 30sec, extension at 72 deg.C, 1min, and circulation for 30 times; 72 ℃ for 10 min; storing at 4 ℃.
5. The method for identifying hevea brasiliensis based on morphological and molecular identification as claimed in claim 2, wherein the PCR reaction is performed using the specific primer C5 (InterspabecetweentnN-GUUandrps 15) of the Chloroplast (Chloroplast) gene of hevea brasiliensis as the detection primer, and the sequence of the primer is as follows: anotation C5
(InterspacebetweentrnN-GUUandrps15)
Forwardprimer:5′-TCAAAGGATCTATGCGCAATCA-3′
Reverseprimer:5′-TCGAGAATTGAAGACCCCTAGT-3′
The total Length of the C5 gene is 462bp, time is 54 ℃,
Enzyme:TaqI,TKS:Polymorphic,TO。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061567A (en) * | 2021-04-06 | 2021-07-02 | 石河子大学 | Method for extracting chloroplast of kochia scoparia |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110275142A1 (en) * | 2010-05-06 | 2011-11-10 | James Wade | Dandelion processes, compositions and products |
| CN111961743A (en) * | 2020-08-24 | 2020-11-20 | 中国热带农业科学院橡胶研究所 | Molecular marker combination for identifying hevea brasiliensis and application thereof |
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- 2021-03-25 CN CN202110321633.2A patent/CN113151418A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110275142A1 (en) * | 2010-05-06 | 2011-11-10 | James Wade | Dandelion processes, compositions and products |
| CN111961743A (en) * | 2020-08-24 | 2020-11-20 | 中国热带农业科学院橡胶研究所 | Molecular marker combination for identifying hevea brasiliensis and application thereof |
Non-Patent Citations (5)
| Title |
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| QUANLIANG XIE 等: ""Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass Taraxacum Kok-saghyz"", 《INT J MOL SCI》 * |
| YINGXIAO ZHANG 等: ""Chloroplast genome resources and molecular markers differentiate rubber dandelion species from weedy relatives"", 《BMC PLANT BIOL》 * |
| ZHANG,Y.,等: ""Taraxacum officinale chloroplast, complete genome"", 《GENBANK》 * |
| ZHANG,Y.等: ""Taraxacum kok-saghyz chloroplast, complete genome"", 《GENBANK》 * |
| 谢全亮 等: ""橡胶草90年来主要研究成果及最新研究进展"", 《植物科学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061567A (en) * | 2021-04-06 | 2021-07-02 | 石河子大学 | Method for extracting chloroplast of kochia scoparia |
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