Primer pair and kit for identifying leaf shape of adiantum nelumboides, and use thereof

Abstract

The present application provides a primer pair and kit for identifying the leaf shape of Adiantum nelumboides, and use thereof. The present application obtains, by means of carrying out sequencing and screening on the genomes of a stably inherited asexual reproduction line "WT" of wild-type Adiantum nelumboides with normal fully rounded leaves and a stably inherited asexual line "BY1" of Adiantum nelumboides with semicircular leaves, a set of loci associated with the leaf shape of Adiantum nelumboides, and designs, on the basis of the loci, an amplification primer pair set forth in SEQ ID NOs: 1-2. This enables the identification of the leaf shape of Adiantum nelumboides at an early stage, improves the accuracy and efficiency of screening, and thus has important theoretical and practical significance.

Description

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

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510354074.3, filed on March 24, 2025, entitled "Primer pairs, reagent kit and application for identifying the shape of Adiantum repens leaf", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a primer pair, reagent kit, and application for identifying the leaf shape of *Adiantum repens*, and relates to the field of molecular breeding technology for forest trees. Background Technology

[0004] *Adiantum reniforme* L. var. *sinese* YXLin 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.

[0005] 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

[0006] This application provides a primer pair for identifying the leaf shape of the Adiantum repens, used to predict leaf shape before the Adiantum repens has grown leaves.

[0007] This application 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.

[0008] The first aspect of this application 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.

[0009] This application involved sequencing and screening 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 were 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 enabled the identification of early leaf shape of maidenhair fern, improving the accuracy and efficiency of screening, and has important theoretical and practical significance.

[0010] The second aspect of this application provides a kit for identifying the leaf shape of *Adiantum repens*, comprising the primer pair provided in the first aspect of this application.

[0011] In one specific embodiment, the kit further includes one or more of DNA extraction reagents, PCR amplification reagents, and amplification product detection reagents.

[0012] Furthermore, in addition to the primers mentioned above, the PCR amplification reagents also include DNA polymerase required for the PCR amplification process.

[0013] Furthermore, the detection reagents for the amplified products can be, for example, the reagents required for PAGE gel electrophoresis detection.

[0014] The third aspect of this application provides the application of the above primer pairs or the above kit in identifying the leaf shape of Adiantum repens.

[0015] In one specific embodiment, Figure 1 is a leaf shape diagram of the Adiantum capillus-veneris provided in an embodiment of this application. As shown in Figure 1, the leaf shape includes a full circular leaf shape as shown in Figure 1A or a semi-circular leaf shape as shown in Figure 1B.

[0016] The fourth aspect of this application provides a method for identifying the shape of the leaves of the Adiantum repens, comprising the following steps:

[0017] DNA was extracted from the Adiantum repens sample to be tested;

[0018] 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;

[0019] 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.

[0020] Based on the primer pair provided in the first aspect of this application, PCR and electrophoresis detection can be performed on the DNA of the sample of Adiantum capillus-veneris to be tested. The leaf shape of the sample of Adiantum capillus-veneris to be tested can be predicted based on the size of the detected fragments, that is, the leaf shape of the sample of Adiantum capillus-veneris to be tested can be predicted to be fully circular or semi-circular.

[0021] In one specific embodiment, the above method includes the following steps:

[0022] Step 1: Extract DNA from the Adiantum hyacinthus sample to be tested.

[0023] 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.

[0024] Genomic DNA was extracted from the sample of Adiantum repens to be tested using conventional techniques in the field, such as the CTAB method.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] This application does not limit how those skilled in the art can perform electrophoretic detection of PCR amplification products, as long as the fragment size of the PCR amplification products can be determined.

[0029] 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.

[0030] This application obtained a set of loci related to the leaf shape of Adiantum repens by sequencing and screening the genomes of the stable genetic asexual line “WT” of wild-type normal full-round leaf Adiantum repens and the stable genetic asexual line “BY1” of semi-round leaf Adiantum repens. Based on these loci, amplification primer pairs as shown in SEQ ID NO:1-2 were designed to identify the early leaf shape of Adiantum repens, which improved the accuracy and efficiency of screening and has important theoretical and practical significance. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 is a diagram of the leaf shape of the maidenhair fern in one embodiment of this application, wherein A is a fully circular leaf shape and B is a semi-circular leaf shape;

[0033] Figure 2 is an electrophoresis result of a semi-circular leaf-shaped maidenhair fern (No. 1) in one embodiment of this application;

[0034] Figure 3 is an electrophoresis result of a semi-circular leaf-shaped maidenhair fern (No. 3) in one embodiment of this application;

[0035] Figure 4 shows the electrophoresis results of a fully circular leaflet of Adiantum repens (No. 6) in one embodiment of this application.

[0036] Figure 5 shows the electrophoresis results of a fully circular leaflet of *Adiantum repens* (No. 8) in one embodiment of this application. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0038] Example 1: Acquisition of primer pairs

[0039] 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 shown in Figure 1. In Figure 1, A is the wild-type normal fully round-leaved maidenhair fern “WT”, and B is the semi-round-leaved maidenhair fern “BY1”. “WT” is the original wild variety of maidenhair fern, which is a stable asexual reproduction line that still maintains the fully round-leaved leaf trait after multiple generations of division and asexual reproduction. “BY1” is an asexual reproduction line that uses wild-type maidenhair fern as the original resource, and produces the semi-round-leaved trait through colchicine mutagenesis, which is stably inherited.

[0040] Step 2, Extraction of whole genome DNA from “WT” and “BY1”:

[0041] ① Take an appropriate amount of tissue (about 200mg fresh weight) and put it into a 2mL 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 60Hz for 180s.

[0042] ② Preparation of CTAB extraction buffer: Weigh 10g CTAB powder, 40.908g NaCl powder, measure 20ml of 0.5mol / L EDTA (pH 8.0), and 50ml of 1mol / L Tris-HCl (pH = 8.0). Finally, adjust the volume to 500mL with ddH2O and store at room temperature.

[0043] ③ Add 750 mL of preheated CTAB extraction buffer (65℃) to the centrifuge tube, shake well to form an emulsion, incubate at 65℃ 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, invert and mix well; centrifuge at 12000 rpm for 5 min at 15℃. Transfer the supernatant to a new centrifuge tube and repeat the previous step.

[0044] 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℃ 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.

[0045] Step 3: Obtaining SSR markers from the whole genome of wild-type Adiantum repens:

[0046] 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.

[0047] Step 4: Mark and filter to obtain:

[0048] 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.

[0049] ②PAGE gel electrophoresis:

[0050] 1) PAGE glue preparation

[0051] 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 (5ml Bind-Silane + 5ml glacial acetic acid + 990ml anhydrous ethanol) to the slab plate and a layer of antisilicide (2ml dimethyldichlorosilane + 98ml anhydrous ethanol) to the concave plate. After the glass plates have dried, place sealing strips on both sides of the slab plate to separate them and secure them with clips. Add 40ml of 6% polyacrylamide gel stock solution, 400μl of 10% ammonium persulfate, and 40μl of TEMED to a small beaker and mix quickly. Slowly pour the solution into the glass through the concave opening. Finally, insert the flat end of the sample comb 0.5cm and secure it with clips. Start electrophoresis after approximately 20-30 minutes.

[0052] 2) Electrophoretic separation

[0053] 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°C for 5 min, then immediately place on ice. Load 4-5 μl of the sample and electrophores at 80 W for 75 min. Stop electrophoresis when the xylene nitrile indicator has passed through 3 / 4 of the plate.

[0054] 3) Silver staining development

[0055] After electrophoresis, remove the plate and pry it open with a knife. Rinse the plate in double-distilled water for a few seconds, then drain off 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 20g NaOH, 0.4g anhydrous Na2CO3, and 2ml 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.

[0056] ③ Mark and filter to obtain

[0057] 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:

[0058] The upstream primer is 5'-TGCGATGTCGAAGTGAGAAC-3' (SEQ ID NO:1)

[0059] The downstream primer is 5'-GATCTTGGCTGCCTCTTTTG-3' (SEQ ID NO:2).

[0060] Example 2: Detection of the leaf shape of the maidenhair fern (Adiantum repens)

[0061] 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:

[0062] ① Take an appropriate amount of tissue (approximately 200 mg fresh weight) and place it in 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 grinder at 60 Hz for 180 s.

[0063] ② Preparation of CTAB extraction buffer: Weigh 10g CTAB powder, 40.908g NaCl powder, measure 20ml of 0.5mol / L EDTA (pH 8.0), and 50ml of 1mol / L Tris-HCl (pH = 8.0). Finally, adjust the volume to 500mL with ddH2O and store at room temperature.

[0064] ③ Add 750 mL of preheated CTAB extraction buffer (65℃) to the centrifuge tube, shake well to form an emulsion, and incubate in a 65℃ 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 rpm for 5 min at 15℃. Transfer the supernatant to a new centrifuge tube and repeat the previous step.

[0065] ④ Transfer the supernatant to a new 1.5 mL centrifuge tube, add 0.7 times the volume of pre-cooled isopropanol, mix by inverting, and incubate at -20°C for 2 hours to allow DNA to precipitate and form a flocculent precipitate. Centrifuge at 12000 rpm for 10 minutes; 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.

[0066] 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 5' end of the upstream primer shown in SEQ ID NO:1 was labeled with the TAMRA fluorescent group; the total PCR reaction volume was 20 μL, including 2 μL DNA template, 0.8 μL upstream and downstream 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.

[0067] Step 3: Perform capillary electrophoresis (CE) on the PCR amplification products to obtain the capillary electrophoresis results, as shown in Figures 2, 3, 4, 5 and Table 1. Figure 2 shows the electrophoresis results for *Adiantum repens* with semi-circular leaves (number 1); Figure 3 shows the results for *Adiantum repens* with semi-circular leaves (number 3); Figure 4 shows the results for *Adiantum repens* with fully circular leaves (number 6); Figure 5 shows the results for *Adiantum repens* with fully circular leaves (number 8); and Table 1 presents the statistical results of the electrophoresis for *Adiantum repens* with semi-circular leaves (numbers 1-5) and fully circular leaves (numbers 6-10).

[0068] Table 1

[0069] As shown in Figures 2, 3, 4, 5, and Table 1, the product size detection results for the semi-circular *Adiantum repens* samples (numbered 1-5) were all within the range of 133 (±1) bp, while the product size detection results for the 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 application 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 in this application 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.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

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.

5. The application according to claim 4, characterized in that, The leaf shape includes a fully circular leaf shape or a semi-circular leaf shape.

6. 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.

7. The method according to claim 6, characterized in that, 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 132-134bp, the leaf of Adiantum repens is identified as semi-circular; when the electrophoresis results show that the PCR amplification product is 126-128bp, the leaf of Adiantum repens is identified as fully circular.

8. The method according to claim 6, characterized in that, The sample of *Adiantum repens* to be tested is at least one of *Adiantum repens* root and *Adiantum repens* 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 Master Mix and 6-7 μL of ddH2O.

10. The method according to claim 6, 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 finally 72℃ extension for 2-8 min.

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