Primer pairs, kits, and applications for identifying the leaf shape of *Adiantum repens*.

Abstract

This invention provides a primer pair, kit, and application for identifying the leaf shape of *Adiantum repens*. The invention involves sequencing and screening the genomes of the stable genetic asexual line “WT” (wild-type, fully circular-leaved) and “BY1” (semi-circular-leaved) of *Adiantum repens*, obtaining a set of loci associated with leaf shape. Based on these loci, amplification primer pairs as shown in SEQ ID NO:1-2 were designed, enabling the identification of early leaf shape in *Adiantum repens*. This improves the accuracy and efficiency of screening and has significant theoretical and practical implications.

Description

Technical Field

[0001] This invention relates to a primer pair, kit, and application for identifying the shape of Adiantum repens leaf blades, and relates to the field of molecular breeding technology for forest trees. Background Technology

[0002] *Adiantum reniforme* L. var. *sinese* YX Lin is a single-leaved evergreen fern belonging to the genus *Adiantum* in the family Adiantaceae. It is endemic to the Three Gorges Reservoir area and is the only plant in the Adiantaceae family found in Asia. Due to its compact size, unique leaf shape, and drought tolerance, *Adiantum reniforme* has strong ornamental value and great market potential in landscaping, indoor foliage cultivation, and ecological restoration.

[0003] The leaf shapes of *Adiantum repens* include fully circular and semi-circular. Currently, the main method for screening *Adiantum repens* with different leaf shapes is to observe them visually after they have grown for a period of time, which is limited by the growth time of *Adiantum repens*. How to predict the leaf shape of *Adiantum repens* before it has grown leaves has attracted the attention of those skilled in the art. Summary of the Invention

[0004] This invention provides a primer pair for identifying the leaf shape of Adiantum repens, used to predict leaf shape before Adiantum repens has grown leaves.

[0005] The present invention also provides a kit including the above primer pairs and the application of the primer pairs and kit in identifying the leaf shape of Adiantum repens.

[0006] The first aspect of the present invention provides a primer pair for identifying the shape of Adiantum repens leaf, comprising a first primer and a second primer, wherein 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] This invention utilizes genome sequencing to screen the genomes of the stable genetic asexual line “WT” of wild-type normal fully circular-leaved maidenhair fern and the stable genetic asexual line “BY1” of semi-circular-leaved maidenhair fern. A set of loci related to the leaf shape of maidenhair fern was obtained, and amplification primer pairs as shown in SEQ ID NO:1-2 were designed based on these loci, where SEQ ID NO:1 is the upstream primer and SEQ ID NO:2 is the downstream primer. This enables the identification of early leaf shape of maidenhair fern, improves the accuracy and efficiency of screening, and has significant theoretical and practical implications.

[0008] A second aspect of the present invention provides a kit for identifying the leaf shape of *Adiantum repens*, comprising the primer pair provided in the first aspect of the present invention.

[0009] In one specific embodiment, the kit further includes one or more of DNA extraction reagents, PCR amplification reagents, and amplification product detection reagents.

[0010] Furthermore, in addition to the primers mentioned above, the PCR amplification reagents also include DNA polymerase required for the PCR amplification process.

[0011] Furthermore, the detection reagents for the amplified products can be, for example, the reagents required for PAGE gel electrophoresis detection.

[0012] A third aspect of the present invention provides the application of the above-mentioned primer pairs or the above-mentioned kit in identifying the leaf shape of Adiantum repens.

[0013] In one specific implementation, Figure 1 A diagram showing the shape of a maidenhair fern leaf according to an embodiment of the present invention, as shown below. Figure 1 As shown, the blade shape includes, for example... Figure 1 The fully circular leaf shape shown in A is or Figure 1 The semi-circular leaf shape shown in B.

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

[0015] DNA was extracted from the Adiantum repens sample to be tested;

[0016] Using the DNA of the Adiantum brevis sample to be tested as a template, PCR amplification was performed using the primer pair provided in the first aspect to obtain PCR amplification products;

[0017] The PCR amplification products were detected by electrophoresis. When the electrophoresis result showed that the PCR amplification product was 132-134 bp, the leaf of the Adiantum capillus-veneris was identified as semi-circular; when the electrophoresis result showed that the PCR amplification product was 126-128 bp, the leaf of the Adiantum capillus-veneris was identified as fully circular.

[0018] According to the primer pair provided in the first aspect of the present invention, the DNA of the sample of Adiantum capillus-veneris to be tested is detected by PCR and electrophoresis. Based on the size of the detected fragments, the leaf shape of the sample of Adiantum capillus-veneris to be tested can be predicted, that is, the leaf of the sample of Adiantum capillus-veneris to be tested is predicted to be fully circular or semi-circular.

[0019] In one specific embodiment, the above method includes the following steps:

[0020] Step 1: Extract DNA from the Adiantum hyacinthus sample to be tested.

[0021] The sample to be tested is any tissue of the maidenhair fern that has not yet grown leaves, and can preferably be at least one of the maidenhair fern roots and stems.

[0022] Genomic DNA was extracted from the sample of Adiantum repens to be tested using conventional techniques in the field, such as the CTAB method.

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

[0024] In the PCR amplification, the PCR reaction system included 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 included: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0025] Step 3: Perform electrophoresis on the PCR amplification products. When the electrophoresis result shows that the PCR amplification product is 132-134 bp, the leaf of the Adiantum capillus-veneris is identified as semi-circular; when the electrophoresis result shows that the PCR amplification product is 126-128 bp, the leaf of the Adiantum capillus-veneris is identified as fully circular.

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

[0027] In one specific embodiment, capillary electrophoresis can be used to detect PCR amplification products. Since capillary electrophoresis requires quantitative analysis based on the intensity of the fluorescence signal, a fluorescent group can be labeled at the 5' end of the first primer. This fluorescent group can be a conventional fluorescent group; in this experiment, the TAMRA fluorescent group was chosen to be labeled at the 5' end of the nucleotide sequence of the first primer.

[0028] This invention utilizes genome sequencing to screen the genomes of the stable genetic asexual line “WT” of wild-type normal fully circular-leaved maidenhair fern and the stable genetic asexual line “BY1” of semi-circular-leaved maidenhair fern. A set of loci related to the leaf shape of maidenhair fern was obtained, and amplification primer pairs as shown in SEQ ID NO:1-2 were designed based on these loci. This enabled the identification of early leaf shape of maidenhair fern, improving the accuracy and efficiency of screening, and has significant theoretical and practical implications. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a diagram showing the leaf shape of a maidenhair fern in one embodiment of the present invention, wherein A is a fully circular leaf shape and B is a semi-circular leaf shape;

[0031] Figure 2 This is an electrophoresis result of a semi-circular leaf-shaped maidenhair fern (number 1) in one embodiment of the present invention.

[0032] Figure 3 This is an electrophoresis result of a semi-circular leaf-shaped maidenhair fern (number 3) in one embodiment of the present invention;

[0033] Figure 4 This is an electrophoresis result of a fully circular leaf-shaped maidenhair fern (number 6) in one embodiment of the present invention.

[0034] Figure 5 This is an electrophoresis result of a fully circular leaflet of Adiantum repens (number 8) in one embodiment of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Example 1: Acquisition of primer pairs

[0037] Step 1: Select the stable genetic asexual reproduction line "WT" of wild-type normal fully round-leaved maidenhair fern and the stable genetic asexual reproduction line "BY1" of semi-round-leaved maidenhair fern. Their leaf shapes are as follows: Figure 1 As shown, Figure 1 A represents the wild-type, normal, fully round-leaved maidenhair fern "WT", while B represents the semi-circular-leaved maidenhair fern "BY1". "WT" is the original wild variety of maidenhair fern, a stable clonal line that retains the fully round-leaved leaf trait after multiple generations of asexual propagation by division. "BY1" is a clonal line that uses wild-type maidenhair fern as the original resource, and produces a semi-circular trait through colchicine mutagenesis, which is stably inherited.

[0038] Step 2, Extraction of whole genome DNA from “WT” and “BY1”:

[0039] ① Take an appropriate amount of tissue (about 200 mg fresh weight) and put it into a 2 mL centrifuge tube. Place it on ice for later use. Add one steel ball to each centrifuge tube and freeze it in liquid nitrogen. Then grind it in a sample grinder at 60 Hz for 180 s.

[0040] ② Preparation of CTAB extraction buffer: Weigh 10 g CTAB powder, 40.908 g NaCl powder, measure 20 ml of 0.5 mol / L EDTA (pH 8.0), and 50 ml of 1 mol / L Tris-HCl (pH = 8.0). Finally, adjust the volume to 500 mL with ddH2O and store at room temperature.

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

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

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

[0044] Based on 13GB of wild-type Adiantum repens transcriptome data, SSR loci were searched, and a total of 79,720 SSR loci were identified. Using published Adiantum repens genome data, based on the principle of balanced distribution of repeat types at each locus, 182 loci were sequenced on the whole genome, and 184 primer pairs were designed using Primer3 software.

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

[0046] ①PCR amplification: Whole-genome DNA extracted from materials “WT” and “BY1” was used for PCR amplification using 184 pairs of primers. The total reaction volume was 20 µL, including 2 µL DNA template, 0.8 µL each of forward and reverse primers, 10 µL 2×Taq Master Mix, and 6.4 µL ddH2O. The PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, for 35 cycles; extension at 72℃ for 5 min.

[0047] ②PAGE gel electrophoresis:

[0048] 1) PAGE glue preparation

[0049] Clean the concave and slab plates used for electrophoresis with detergent, rinse with tap water, and let them air dry on a rack. Wipe them clean with anhydrous ethanol. Apply a layer of silicide (5 ml Bind-Silane + 5 ml glacial acetic acid + 990 ml anhydrous ethanol) to the slab plate and a layer of antisilicide (2 ml dimethyldichlorosilane + 98 ml anhydrous ethanol) to the concave plate. After the glass plates have dried, place sealing strips on both sides of the slab plate to separate the glass plates and secure them with clips. Add 40 ml of 6% polyacrylamide gel stock solution, 400 µl of 10% ammonium persulfate, and 40 µl of TEMED to a small beaker and mix quickly. Slowly pour the solution into the glass through the concave opening, and finally insert the flat end of the sample comb 0.5 cm and secure it with clips. Start electrophoresis after approximately 20-30 minutes.

[0050] 2) Electrophoretic separation

[0051] Remove the comb from the solidified plate and carefully clean the gel fragments at the gel inlet under tap water. Fix the glass plate onto the electrophoresis tank and add 0.5× TBE buffer. Preheat the electrophoresis tank for 20 min, set the electrophoresis voltage to 2000 V, current to 100 mA, and power to 80 W. After electrophoresis, insert the comb. Add an equal volume of loading buffer (98% deionized formamide, 10 mmol / L EDTA, 0.005% xylene nitrile, 0.005% bromophenol blue) to the selectively amplified PCR products. Denature at 95℃ for 5 min, then immediately place on ice. Load 4-5 µl of the sample and electrophore at 80 W for 75 min. Stop electrophoresis when the xylene nitrile indicator has passed 3 / 4 of the plate.

[0052] 3) Silver staining development

[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, then remove and drain the surface water. Transfer it to silver staining solution (0.18% AgNO3) and shake slowly on a shaker. After silver staining for 10-20 minutes, remove the plate and remove as much silver staining solution as possible from the surface. Transfer it to developing solution (containing 20 g NaOH, 0.4 g anhydrous Na2CO3, and 2 ml formaldehyde per liter of water) and shake on a shaker for 5-10 minutes until the bands are clearly visible. Remove the plate, rinse it thoroughly with running water, and air dry. Record the bands and photograph them for preservation.

[0054] ③ Mark and filter to obtain

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

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

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

[0058] Example 2: Detection of the leaf shape of the maidenhair fern (Adiantum repens)

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

[0060] ① Take an appropriate amount of tissue (approximately 200 mg fresh weight) and place it in a 2 mL centrifuge tube. Place the tube on ice for later use. Add one steel ball to each centrifuge tube and freeze in liquid nitrogen. Then grind the tissue in a grinder at 60 Hz for 180 s.

[0061] ② Preparation of CTAB extraction buffer: Weigh 10 g CTAB powder, 40.908 g NaCl powder, measure 20 ml of 0.5 mol / L EDTA (pH 8.0), and 50 ml of 1 mol / L Tris-HCl (pH = 8.0). Finally, adjust the volume to 500 mL with ddH2O and store at room temperature.

[0062] ③ Add 750 mL of preheated CTAB extraction buffer (65°C) to the centrifuge tube, shake well to form an emulsion, and incubate in a 65°C water bath for 45 min, shaking several times during incubation. Remove and cool to room temperature, then add an equal volume of chloroform-isoamyl alcohol (24:1) mixture, and mix by inverting the tube. Centrifuge at 12000 r / min for 5 min at 15°C. Transfer the supernatant to a new centrifuge tube and repeat the previous step.

[0063] ④ Transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.7 times the volume of pre-chilled isopropanol, mix by inverting, and incubate at -20°C for 2 h to allow DNA to precipitate and aggregate into a flocculent precipitate. Centrifuge at 12000 r / min for 10 min; discard the supernatant, wash twice with 75% ethanol and once with anhydrous ethanol, air dry on a clean bench, and dissolve in 50 μl of ddH2O to obtain genomic DNA.

[0064] Step 2, PCR amplification. Using the DNA from the Adiantum repens sample to be tested as a template, PCR was performed using the upstream and downstream primers shown in SEQ ID NO:1-2. The upstream primer shown in SEQ ID NO:1 was labeled with the TAMRA fluorescent group at its 5' end. The total PCR reaction volume 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 as follows: pre-denaturation at 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, for 35 cycles; extension at 72℃ for 5 min.

[0065] Step 3: Perform capillary electrophoresis (CE) on the PCR amplification products to obtain the capillary electrophoresis results, which can be found in the attached image. Figure 2 , Figure 3 , Figure 4 , Figure 5 And Table 1. Among them, Figure 2 This is an electrophoresis result of the semi-circular leaf-shaped maidenhair fern (number 1). Figure 3 This is an electrophoresis result of the semi-circular leaf-shaped maidenhair fern (number 3). Figure 4 This is an electrophoresis result of the Adiantum repens (number 6), a fern with fully circular leaves. Figure 5 Table 1 shows the electrophoresis results of Adiantum repens with fully circular leaves (number 8). Table 1 shows the electrophoresis statistics of Adiantum repens with semi-circular leaves (numbers 1-5) and Adiantum repens with fully circular leaves (numbers 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 for semi-circular *Adiantum repens* samples (numbered 1-5) were all within the range of 133 (±1) bp, while the product size detection results for fully circular *Adiantum repens* samples (numbered 6-10) were all within the range of 127 (±1) bp. These ranges are due to fluctuations in capillary electrophoresis peak readings. These results indicate that the amplification products obtained using the primer pairs of this invention can distinguish between fully circular and semi-circular leaf shapes in *Adiantum repens*. When the amplification product size is 133 (±1) bp, the leaf shape is semi-circular; when the amplification product size is 127 (±1) bp, the leaf shape is fully circular. Therefore, the primer pairs provided by this invention can accurately identify the leaf shape of *Adiantum repens*, without being limited by the growth time of *Adiantum repens*, and can predict the leaf shape even before the leaves have grown.

[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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above 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 shape of Adiantum repens leaf blades, characterized in that, It includes 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.

2. A kit for identifying the leaf shape of *Adiantum repens*, characterized in that, Includes the primer pair as described in claim 1.

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

4. The use of the primer pair of claim 1 or the kit of any one of claims 2-3 in identifying the leaf shape of Adiantum repens; The leaf shape includes a fully circular leaf shape or a semi-circular leaf shape.

5. A method for identifying the shape of the leaves of the *Adiantum repens*, characterized in that, Includes the following steps: DNA was extracted from the Adiantum repens sample to be tested; Using the DNA of the Adiantum lancifolium sample to be tested as a template, PCR amplification was performed using the primer pair described in claim 1 to obtain the PCR amplification product; The PCR amplification products were subjected to electrophoresis detection, and the leaf shape of the Adiantum repens was identified based on the electrophoresis results. The method of identifying the leaf shape of Adiantum repens based on electrophoresis results also includes: when the electrophoresis results show that the PCR amplification product is 132-134 bp, the leaf of Adiantum repens is identified as semi-circular. When the electrophoresis results show that the PCR amplification product is 126-128 bp, the leaf of the Adiantum capillus-veneris is identified as being fully circular.

6. The method according to claim 5, characterized in that, The sample of *Adiantum repens* to be tested is at least one of *Adiantum repens* root and *Adiantum repens* stem.

7. The method according to claim 5, 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.

8. The method according to claim 5, characterized in that, The PCR amplification program includes: 95℃ pre-denaturation for 4-6 min; 95℃ denaturation for 25-35 s, 56℃ annealing for 25-35 s, 72℃ extension for 25-35 s, for a total of 35 cycles; and a final extension at 72℃ for 2-8 min.

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