Primer pair and kit for identifying leaf shape of adiantum obtusifolius and application of primer pair and kit

Abstract

The invention provides a primer pair and a kit for identifying the leaf shape of adiantum obtusifolius and application of the primer pair and the kit. According to the invention, genomes of a stable genetic asexual reproduction line 'WT' and a stable genetic asexual reproduction line 'BY1' of wild normal full-round leaf adiantum lotus leaf are subjected to sequencing screening, a group of sites related to leaf shapes of adiantum lotus leaf are obtained, and an amplification primer pair shown as SEQ ID NO: 1-2 is designed based on the sites; the method realizes the identification of the shape of the early leaf of the adiantum lotus, improves the screening accuracy and efficiency, and has important theoretical and practical significance.

Description

Technical Field

[0001] The invention relates to a primer pair, a kit and an application for identifying the leaf shape of Adiantum neriifolia, and relates to the technical field of forest tree molecular breeding. Background Art

[0002] Adiantum reniforme L. var.sinese YX Lin is a simple-leaf evergreen plant of the genus Adiantum L. of the family Adiantaceae. It is a species endemic to the Three Gorges Reservoir area and the only plant of the family Adiantum distributed in Asia. Adiantum reniforme has a small plant shape, unique leaf shape, certain drought tolerance, and strong ornamental value. It has great market application prospects in garden plant landscaping, indoor foliage viewing, and ecological restoration applications.

[0003] The leaf shapes of Adiantum sibiricum include full circle and semicircle. At present, the method of screening Adiantum sibiricum with different leaf shapes is mainly to wait for a period of growth and then identify them by naked eye observation, which is limited by the growth time of Adiantum sibiricum. How to predict the leaf shape of Adiantum sibiricum before it grows leaves has attracted the attention of technicians in this field. Summary of the invention

[0004] The invention provides a primer pair for identifying the leaf shape of Adiantum cyrtonema, which is used for predicting the leaf shape at the stage when the Adiantum cyrtonema has not yet grown leaves.

[0005] The present invention also provides a kit comprising the primer pair and application of the primer pair and the kit in identifying the leaf shape of Adiantum nymphaeaceae.

[0006] A first aspect of the present invention provides a primer pair for identifying the leaf shape of Adiantum nudibranch, comprising a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:1, and the nucleotide sequence of the second primer is shown in SEQ ID NO:2.

[0007] The present invention obtains a group of sites related to the leaf shape of Adiantum nymphaeaceae by sequencing and screening the genomes of a wild-type normal fully circular leaf Adiantum nymphaeaceae stable genetic asexual reproduction line "WT" and a semicircular leaf Adiantum nymphaeaceae stable genetic asexual reproduction line "BY1", and designs an amplification primer pair as shown in SEQ ID NO:1-2 based on the sites, wherein SEQ ID NO:1 is an upstream primer and SEQ ID NO:2 is a downstream primer, thereby realizing the identification of the early leaf shape of Adiantum nymphaeaceae, improving the accuracy and efficiency of screening, and having important theoretical and practical significance.

[0008] The second aspect of the present invention provides a kit for identifying the leaf shape of Adiantum nymphaeaceae, comprising the primer pair provided by the first aspect of the present invention.

[0009] In a specific embodiment, the kit further comprises one or more of a DNA extraction reagent, a PCR amplification reagent, and an amplification product detection reagent.

[0010] Furthermore, the PCR amplification reagent includes, in addition to the above primer pairs, a DNA polymerase required for the PCR amplification process.

[0011] Furthermore, the amplification product detection reagent may be, for example, a reagent required for PAGE gel electrophoresis detection.

[0012] The third aspect of the present invention provides the use of the above primer pair or the above kit in identifying the leaf shape of Adiantum nudiflorum.

[0013] In a specific embodiment, Figure 1 The shape diagram of the leaves of the lotus leaf fern provided by an embodiment of the present invention is as follows: Figure 1 As shown, the blade shape includes Figure 1 The fully round leaf shape shown in A or Figure 1 The semicircular leaf shape shown in B.

[0014] A fourth aspect of the present invention provides a method for identifying the shape of Adiantum officinale leaves, comprising the following steps:

[0015] Extracting DNA from the Adiantum oleifera sample to be tested;

[0016] Using the DNA of the Adiantum nerii sample to be detected as a template, PCR amplification is performed using the primer pair provided in the first aspect to obtain a PCR amplification product;

[0017] The PCR amplification product is subjected to electrophoresis detection. When the electrophoresis result shows that the PCR amplification product is 132-134 bp, the leaf of Adiantum palmatum is identified as semicircular; when the electrophoresis result shows that the PCR amplification product is 126-128 bp, the leaf of Adiantum palmatum is identified as fully circular.

[0018] According to the primer pairs provided in the first aspect of the present invention, PCR detection and electrophoresis detection are performed on the DNA of the Adiantum sample to be detected, and the leaf shape of the Adiantum sample to be detected can be predicted based on the size of the fragments appearing in the detection, that is, it is predicted that the leaves of the Adiantum sample to be detected are fully circular or semi-circular.

[0019] In a specific embodiment, the above method comprises the following steps:

[0020] Step 1: extract the DNA of the Adiantum nerii sample to be tested.

[0021] The sample of Adiantum to be tested refers to any tissue of Adiantum that has not grown leaves, and can preferably be at least one of the root of Adiantum and the stem of Adiantum.

[0022] The genomic DNA of the Adiantum nymphaeaceae sample to be tested is extracted by conventional technical means in the art, for example, the CTAB method can be used.

[0023] Step 2: Using the DNA of the Adiantum nerii sample to be detected as a template, perform PCR amplification using the primer pair described in claim 1 to obtain a PCR amplification product.

[0024] In the PCR amplification, the PCR reaction system includes 2 µL of DNA template, 0.8 µL of the first primer, 0.8 µL of the second primer, 10 µL of 2×Taq Master Mix and 6.4 µL of ddH2O. The PCR amplification program includes: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, a total of 35 cycles; and finally 72°C extension for 5 min.

[0025] Step 3, performing electrophoresis detection on the PCR amplification product, when the electrophoresis result shows that the PCR amplification product is 132-134 bp, the leaf of Adiantum lotus leaf is identified as semicircular; when the electrophoresis result shows that the PCR amplification product is 126-128 bp, the leaf of Adiantum lotus leaf is identified as fully circular.

[0026] The present invention does not limit how those skilled in the art perform electrophoresis detection of PCR amplification products, as long as the fragment size of the PCR amplification product can be determined.

[0027] In a specific embodiment, capillary electrophoresis can be used to detect PCR amplification products. Since capillary electrophoresis requires quantitative analysis by the intensity of the fluorescent signal, a fluorescent group can be labeled at the 5' end of the first primer, and the fluorescent group can be a conventional fluorescent group. In this experiment, the TAMRA fluorescent group is selected to be labeled at the 5' end of the nucleotide sequence of the first primer.

[0028] The present invention obtains a group of sites related to the leaf shape of Adiantum nymphaeaceae by sequencing and screening the genomes of a wild-type normal fully circular leaf Adiantum nymphaeaceae stable genetic asexual reproduction line "WT" and a semicircular leaf Adiantum nymphaeaceae stable genetic asexual reproduction line "BY1", and designs amplification primer pairs as shown in SEQ ID NO:1-2 based on the sites, thereby realizing the identification of the early leaf shape of Adiantum nymphaeaceae, improving the accuracy and efficiency of screening, and having important theoretical and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 The shape diagram of the leaves of Adiantum oleifera in one embodiment of the present invention, wherein A is a fully circular leaf shape, and B is a semicircular leaf shape;

[0031] Figure 2 This is a graph showing the electrophoresis result of Adiantum officinale (No. 1) with semicircular leaves in one embodiment of the present invention;

[0032] Figure 3 This is a graph showing the electrophoresis result of Adiantum officinale (No. 3) with semicircular leaves in one embodiment of the present invention;

[0033] Figure 4 This is a graph showing the electrophoresis result of Adiantum officinale (No. 6) with fully circular leaves in one embodiment of the present invention;

[0034] Figure 5 This is a diagram showing the electrophoresis results of the fully circular leaves of the lotus leaf fern (No. 8) in one embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1: Acquisition of primer pairs

[0037] Step 1: Select the wild-type normal full-circular leaf lotus maidenhair fern stable genetic asexual reproduction line "WT" and the semi-circular leaf lotus maidenhair fern stable genetic asexual reproduction line "BY1", whose leaf shape is as follows Figure 1 As shown, Figure 1 A in the middle is the wild-type normal fully circular leaf Adiantum "WT", and B is the semi-circular leaf Adiantum "BY1"; "WT" is the wild original variety of Adiantum, which is a stable asexual line that still maintains the fully circular leaf trait after many generations of asexual reproduction; "BY1" is an asexual line that uses the wild-type Adiantum as the original resource, and produces the semi-circular trait after colchicine mutagenesis, and is stably inherited.

[0038] Step 2, "WT" and "BY1" whole genome DNA extraction:

[0039] ① Take an appropriate amount of tissue (about 200 mg fresh weight) and put it into a 2 mL centrifuge tube and place it on ice for later use; add a steel ball to each centrifuge tube and freeze it in liquid nitrogen; then grind it in a sampler at 60 Hz and 180 s;

[0040] ② Preparation of CTAB extraction buffer: weigh 10 g CTAB powder, 40.908 g NaCl powder, 20 ml 0.5 mol / L EDTA (pH 8.0), 50 ml 1 mol / L Tris-HCl (pH = 8.0), and finally dilute to 500 mL with ddH2O and store at room temperature;

[0041] ③ Add 750 mL of 65°C preheated CTAB extraction buffer to the centrifuge tube, shake and mix until it becomes milky, and place in a 65°C water bath for 45 min, shaking and mixing several times during the process; take out and cool to room temperature, add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, and mix by inverting; centrifuge at 15°C 12000 r / min for 5 min. Transfer the supernatant to a new centrifuge tube and repeat the previous step;

[0042] ④ Take the supernatant and put it into a new 1.5 mL centrifuge tube. Add 0.7 times the volume of pre-cooled isopropanol, mix it upside down, and place it at -20°C for 2 h to allow the DNA to precipitate into flocculent sediment; centrifuge it at 12000 r / min for 10 min; discard the supernatant, wash it twice with 75% ethanol and once with anhydrous ethanol, air-dry it on a clean bench, and dissolve it with 50 μl of ddH2O after air-drying to obtain genomic DNA.

[0043] Step 3: Obtaining SSR markers of the whole genome of wild-type Adiantum spheniscus:

[0044] Based on 13GB of wild-type Adiantum lily transcriptome data, SSR loci were searched and a total of 79,720 SSR loci were identified. Using the published Adiantum lily genome data and based on the principle of balanced distribution of repeat types at each locus, 182 loci were screened and sequenced on the whole genome, and 184 pairs of primers were designed using primer3 software.

[0045] Step 4: Mark and filter to obtain:

[0046] ① PCR amplification: The whole genome DNA extracted from "WT" and "BY1" materials was selected and PCR amplified using the 184 pairs of primers designed. The total reaction system was 20 µL, including 2 µL DNA template, 0.8 µL forward and reverse primers, 10 µL 2×Taq Master Mix, and 6.4 µL ddH2O; the PCR amplification program was: pre-denaturation at 95℃ for 5min; 95℃ for 30s, 56℃ for 30s, and 72℃ for 30s, 35 cycles; extension at 72℃ for 5min;

[0047] ②PAGE gel electrophoresis:

[0048] 1) PAGE gel preparation

[0049] Clean the gravure plate and plate used for electrophoresis with detergent, then rinse with tap water and place on a stand to dry. Clean with anhydrous ethanol, apply a layer of siliconizing agent (5 ml Bind-Silane + 5 ml glacial acetic acid + 990 ml anhydrous ethanol) on the plate, and apply a layer of anti-siliconizing agent (2 ml dimethyldichlorosilane + 98 ml anhydrous ethanol) on the gravure plate. After the glass plate is left to dry, place seals on both sides of the plate to isolate the glass plate and fix it with a clamp. Add 40 ml of 6% polyacrylamide gel master solution, 400 µl of 10% ammonium persulfate and 40 µl TEMED to a small beaker and mix quickly. Slowly pour in from the glass notch, and finally insert the flat end of the spotting comb 0.5 cm and fix it with a clamp. Start electrophoresis after about 20-30 minutes;

[0050] 2) Electrophoretic separation

[0051] Remove the comb from the condensed plate and carefully clean the broken gel at the gel mouth under running water. Fix the glass plate to the electrophoresis tank and add 0.5× TBE buffer. Preheat the electrophoresis for 20 min, with an electrophoresis voltage of 2000 v, a current of 100 mA, and a power of 80 W. Insert the comb after the electrophoresis is completed. Add an equal volume of loading buffer (98% deionized formamide, 10 mmol / L EDTA, 0.005% xylene cyanol, 0.005% bromophenol blue) to the selectively amplified PCR product. Denature at 95°C for 5 min and immediately place on ice, load 4-5 µl, and run the electrophoresis at 80 W for 75 min. Stop the electrophoresis when the xylene cyanol indicator runs over 3 / 4 of the plate.

[0052] 3) Silver staining

[0053] After electrophoresis, remove the plate and pry open the intaglio plate with a knife. Rinse the plate in double distilled water for a few seconds, remove and drain the water on the surface. Then transfer to silver stain solution (0.18% AgNO3) and shake slowly on a shaker. After 10-20 minutes of silver staining, remove and try to remove the silver stain solution on the surface, transfer to the developer (containing 20 g NaOH, 0.4 g anhydrous Na2CO3, 2 ml formaldehyde per liter of water), and shake on a shaker for 5-10 minutes until the bands are clearly visible, remove and rinse with running water, dry, record the bands and take photos for preservation.

[0054] ③ Marker screening acquisition

[0055] According to the detection results of 184 pairs of primer amplification electrophoresis, primer pairs with suitable length, matching annealing temperature, low background noise and good specificity were screened, including upstream primers and downstream primers:

[0056] The upstream primer is 5'-TGCGATGTCGAAGTGAGAAC-3' (SEQ ID NO: 1)

[0057] The downstream primer was 5'-GATCTTGGCTGCCTCTTTTG-3' (SEQ ID NO: 2).

[0058] Example 2: Detection of the shape of the leaves of Adiantum sibiricum to be detected

[0059] Step 1: Using the CTAB method, genomic DNA was extracted from 5 Adiantum spp. with semicircular leaves (numbered 1-5) and 5 Adiantum spp. with fully circular leaves (numbered 6-10). The specific steps include:

[0060] ① Take an appropriate amount of tissue (about 200 mg fresh weight) and put it into a 2 mL centrifuge tube and place it on ice for later use. Add a steel ball to each centrifuge tube and freeze it in liquid nitrogen; then grind it in a sampler at 60 Hz and 180 s;

[0061] ② Preparation of CTAB extraction buffer: weigh 10 g CTAB powder, 40.908 g NaCl powder, 20 ml 0.5 mol / L EDTA (pH 8.0), 50 ml 1 mol / L Tris-HCl (pH = 8.0), and finally dilute to 500 mL with ddH2O and store at room temperature;

[0062] ③ Add 750 mL of 65°C preheated CTAB extraction buffer to the centrifuge tube, shake and mix until it becomes milky, and place in a 65°C water bath for 45 min, shaking and mixing several times during the process. Take out and cool to room temperature, add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, and mix by inverting. Centrifuge at 15°C 12000 r / min for 5 min. Transfer the supernatant to a new centrifuge tube and repeat the previous step;

[0063] ④ Take the supernatant and put it in a new 1.5 mL centrifuge tube. Add 0.7 times the volume of pre-cooled isopropanol, mix it upside down, and place it at -20°C for 2 h to allow the DNA to precipitate into flocculent precipitates. Centrifuge at 12000 r / min for 10 min; discard the supernatant, wash it twice with 75% ethanol and once with anhydrous ethanol, air-dry it on a clean bench, and dissolve it with 50 μl of ddH2O after air-drying to obtain genomic DNA.

[0064] Step 2, PCR amplification. The DNA of the sample of Adiantum oleifera to be detected was used as a template, and PCR was performed using the upstream and downstream primers shown in SEQ ID NO:1-2 respectively; wherein the 5' end of the upstream primer shown in SEQ ID NO:1 was labeled with a TAMRA fluorescent group; the total PCR reaction system was 20 µL, including 2 µL of DNA template, 0.8 µL of upstream and downstream primers, 10 µL of 2×Taq Master Mix, and 6.4 µL of ddH2O; the PCR amplification program was: pre-denaturation at 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s, after 35 cycles; extension at 72°C for 5 min.

[0065] Step 3: Perform capillary electrophoresis (CE) on the PCR amplification product to obtain the capillary electrophoresis results. Figure 2 , Figure 3 , Figure 4 , Figure 5 and Table 1. Among them, Figure 2 This is the electrophoresis result of the semicircular leaves of the lotus leaf fern (No. 1). Figure 3 This is the electrophoresis result of the semicircular leaves of the lotus leaf fern (No. 3). Figure 4 This is the electrophoresis result of the completely round leaves of the lotus leaf fern (No. 6). Figure 5 Figure 1 is the electrophoresis result of the fully circular leaves of Adiantum officinale (No. 8), and Table 1 is the electrophoresis statistical results of the semicircular leaves of Adiantum officinale (No. 1-5) and the fully circular leaves of Adiantum officinale (No. 6-10).

[0066] Table 1

[0067]

[0068] like Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown in Table 1, the product size detection results of the semicircular lotus leaf Adiantum samples (numbered 1-5) are all within the result range of 133 (± 1) bp, and the product size detection results of the fully circular lotus leaf Adiantum samples (numbered 6-10) are all within the result range of 127 (± 1) bp; the result range here is due to a certain fluctuation generated when the capillary electrophoresis peak reading is read. The above results show that the amplified product obtained using the primer pair of the present invention can distinguish whether the shape of the lotus leaf Adiantum leaf is a fully circular leaf shape or a semicircular leaf shape. When the size of the amplified product is 133 (± 1) bp, the shape of the lotus leaf Adiantum leaf is a semicircular leaf shape; when the size of the amplified product is 127 (± 1) bp, the shape of the lotus leaf Adiantum leaf is a fully circular leaf shape. Therefore, the primer pair provided by the present invention can accurately identify the leaf shape of the lotus leaf Adiantum leaf, will not be limited by the growth time of the lotus leaf Adiantum leaf, and can predict the leaf shape at the stage when the lotus leaf Adiantum leaf has not yet grown leaves.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer pair for identifying the leaf shape of Adiantum nymphaeaceae, characterized in that: It comprises a first primer and a second primer, wherein the nucleotide sequence of the first primer is shown as SEQ ID NO:1, and the nucleotide sequence of the second primer is shown as SEQ ID NO:

2.

2. A kit for identifying the leaf shape of Adiantum nymphaeaceae, characterized in that: Comprising the primer pair described in claim 1.

3. The kit according to claim 2, characterized in that The kit also includes a DNA polymerase.

4. Use of the primer pair according to claim 1 or the kit according to any one of claims 2 to 3 in identifying the leaf shape of Adiantum nudiflorum.

5. The use according to claim 4, characterized in that: The blade shape includes a full-circular blade shape or a semi-circular blade shape.

6. A method for identifying the shape of the leaves of Adiantum scabra, characterized in that: The steps include: Extracting DNA from the Adiantum oleifera sample to be tested; Using the DNA of the Adiantum nerii sample to be detected as a template, PCR amplification is performed using the primer pair described in claim 1 to obtain a PCR amplification product; The PCR amplification product is subjected to electrophoresis detection, and the leaf shape of Adiantum nucifera is identified according to the electrophoresis result.

7. The method according to claim 6, characterized in that The identifying of the shape of the Adiantum officinale leaves according to the electrophoresis results also includes: when the electrophoresis results show that the PCR amplification product is 132-134 bp, identifying the Adiantum officinale leaves as semicircular; when the electrophoresis results show that the PCR amplification product is 126-128 bp, identifying the Adiantum officinale leaves as fully circular.

8. The method according to claim 6, characterized in that The Adiantum officinale sample to be detected is at least one of Adiantum officinale root and Adiantum officinale stem.

9. The method according to claim 6, characterized in that In the PCR amplification, the PCR reaction system includes 1-3 µL of DNA template, 0.5-1.0 µL of the first primer, 0.5-1.0 µL of the second primer, 8-12 µL of 2×Taq MasterMix and 6-7 µL of ddH2O.

10. The method according to claim 6, characterized in that In the PCR amplification, the PCR amplification program includes: pre-denaturation at 95°C for 4-6 min; denaturation at 95°C for 25-35 s, annealing at 56°C for 25-35 s, and extension at 72°C for 25-35 s, for a total of 35 cycles; and finally extension at 72°C for 2-8 min.

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