A fragment, primer, kit and method for sex identification of Nepenthes

Abstract

The present invention discloses a fragment, primer, kit, and method for sex identification of Nepenthes. The method utilizes genome-wide association analysis to identify the sex-linked region of the Nepenthes Y chromosome, further screening for male-specific fragments. Primers for specific molecular markers are designed using these fragments. The primers include a forward primer and a reaction primer. PCR amplification is performed on extracted Nepenthes DNA to obtain an amplified product, which is then subjected to agarose gel electrophoresis to generate distinct bands that are organized into a fingerprint pattern. A stable and distinct amplification result is obtained, allowing Nepenthes of different sexes to be distinguished. The method is used for early sex identification of Nepenthes, exhibits excellent stability, is applicable to different Nepenthes individuals, and is of great significance for sex identification of Nepenthes.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a fragment, a primer, a kit and a method for sex identification of a pitcher plant. Background Art

[0002] Nepenthes Nepenthes mirabilis ) is a climbing herb with lanceolate leaves. Pitchers extend from the midrib at the top of the leaves to differentiate into insect-catching pitchers. Due to the unique morphology and predatory nature of Nepenthes, it has extremely high scientific and ornamental value. Nepenthes flowers are unisexual and are dioecious plants. When the male flowers bloom, they emit a special smell to attract insects for pollination. Nepenthes has two reproductive methods: vegetative reproduction and seed reproduction, but the germination rate of its seeds in the wild is extremely low. Therefore, in the wild, Nepenthes mainly reproduces vegetatively by creeping branches on the ground and taking root. At present, the identification of Nepenthes sex mainly depends on the differentiation of the male and female flower morphology of the reproductive organs, such as Figure 1 As shown, sex identification is impossible in the early stages of a young plant, before the male and female flowers differentiate. This inability to identify sex in the early stages significantly hinders field surveys and sampling of pitcher plants, as well as their horticultural breeding, cultivation, and artificial hybridization.

[0003] In previous research reports, Scharmann et al. (2019) performed double enzyme restriction site sequencing (ddRAD-seq) on three Nepenthes species and found the male-specific site of Nepenthes. They then conducted a study on Nepenthes indica ( N. khasiana ) by sequencing and assembling the transcriptome of the male inflorescence of Nepenthes spp., identifying a male-specific gene (DYT1) and developing a corresponding molecular marker: primers F: 5'-AATTCACTGATTCGGATCACG-3'; R: 5'-CGATCGCGTCGCAAAGTATG-3'. The mitochondrial gene cox1, shared by both male and female Nepenthes plants, was used as an internal reference gene (primers F: 5'-GGAGGAGTTGATTTAGC-3'; R: 5'-AAGGCTGGAGGGCTTTGTAC-3'). PCR-based experiments confirmed the specificity of the DYT1 gene in male Nepenthes plants. However, this study has certain limitations. For example, the double-enzyme restriction site sequencing method provided low genome coverage, potentially yielding incomplete information on the DYT1 gene. Furthermore, the PCR product generated by the primers developed for DYT1 was relatively short, resulting in unclear results on agarose gel electrophoresis. In addition, in this study, the number of experimental individuals of the same species was small, and the stability of the molecular marker was lacking and needed further verification. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a fragment, primers, a kit and a method for sex identification of Nepenthes.

[0005] The technical solution is as follows: In a first aspect, the present invention provides a fragment for identifying the sex of a pitcher plant, and the nucleotide sequence of the fragment is shown as SEQ ID NO.1.

[0006] The second aspect of the present invention provides a primer for identifying sex-specific molecular markers of Nepenthes, wherein the primer is designed based on the nucleotide sequence of the fragment described in SEQ ID NO.1.

[0007] Preferably, the primers include a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.3.

[0008] The invention relates to the use of the primers described in the second aspect in sex identification of Nepenthes.

[0009] A third aspect of the present invention provides a kit for identifying the sex of Nepenthes, comprising the primers described in the second aspect of the present invention.

[0010] A fourth aspect of the present invention provides a method for identifying the sex of a pitcher plant, comprising the following steps:

[0011] 1) Extract DNA from the pitcher plant to be identified;

[0012] 2) using DNA of the pitcher plant to be identified as a template and performing PCR amplification using the primers described in claim 3 or claim 4 or the kit described in claim 5;

[0013] 3) Detect PCR amplification products. When the amplification products show:

[0014] When the 523 bp band is detected, the pitcher plant to be identified is a male pitcher plant;

[0015] Alternatively, when the amplified product does not show a 523 bp band, the pitcher plant to be identified is a female pitcher plant.

[0016] Preferably, in step 2), the reaction system for PCR amplification is: a total reaction system of 15 μl, including 7.5 μl of DNA polymerase, 1 μl of forward primer, 1 μl of reverse primer, 1 μl of DNA of the pitcher plant to be identified, and 4.5 μl of sterile water, wherein the nucleotide sequences of the forward primer and the reverse primer are shown as SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0017] Preferably, the PCR reaction condition program includes: 95°C, pre-denaturation of the DNA of the pitcher plant to be identified for 5 minutes; 95°C, denaturation for 1 minute; 55°C, annealing for 1 minute; 72°C, extension for 1 minute; wherein the denaturation-annealing-extension program is repeated 34 times, and then fully extended at 72°C for 5 minutes.

[0018] Compared with the existing technology, the present invention has the following beneficial effects: the present invention uses whole-genome association analysis to lock the sex-linked region of the Y chromosome of Nepenthes, and further screens male-specific fragments. Targeting the male-specific fragments of Nepenthes, primers, kits and methods for molecular markers are developed, and molecular experiments are optimized for early sex identification of Nepenthes. The identification method has excellent stability and is applicable to different individuals of Nepenthes, which is of great significance for sex identification of Nepenthes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a characteristic diagram of male and female pitcher plant inflorescences;

[0020] Figure 2 The figure shows the PCR amplification bands. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand. Example

[0022] To obtain specific gene sequences, based on third-generation sequencing of male and female Nepenthes plants, complete reference genomes for both sexes were obtained. Deep resequencing was performed on 80 different Nepenthes individuals, and genome-wide association studies (GWAS) were performed to identify Y chromosome linkage regions. Transcriptome sequencing of male and female Nepenthes floral organs was further performed, and differential expression and enrichment analyses were performed to compare molecular differences between male and female individuals. This revealed a male-specific fragment, designated NM-MSG. Bioinformatics methods and data comparison confirmed that the NM-MSG sequence differed from the previously identified DYT1 gene. Since female Nepenthes plants do not contain this fragment, primers based on this fragment were designed to further identify the sex of the Nepenthes plants.

[0023] The nucleotide sequence of NM-MSG is shown in SEQ ID NO.1:

[0024]

[0025] Primers were designed based on the specific fragment, which is unique to male pitcher plants. After obtaining the sequence of the specific fragment NM-MSG, primers were designed using Primer3plus (https: / / www.primer3plus.com). The specificity of the primers was initially verified using NCBI blast. The primers can amplify the male-specific fragment via PCR. The primers designed to obtain NM-MSG include a forward primer and a reverse primer.

[0026] The nucleotide sequence of the forward primer is shown in SEQ ID NO. 2: 5′-TTCTGCCCAGCTCAATGGAG-3′;

[0027] The nucleotide sequence of the reverse primer is shown in SEQ ID NO. 3: 5'-CCGGAAGACAGCATCACAGT-3'.

[0028] The mitochondrial gene cox1, shared by both male and female pitcher plants, was used as an internal reference gene. Under exactly the same conditions, the primers for the internal reference gene could amplify gene sequences in both male and female individuals, while the primers for NM-MSG could only amplify gene sequences in male individuals but not in females.

[0029] The primers for the internal reference gene cox1 are: forward primer (F): 5'-GGAGGAGTTGATTTAGC-3';

[0030] Reverse primer (R): 5′-AAGGCTGGAGGGCTTTGTAC-3′.

[0031] The above-designed NM-MSG primers and the primer sequences of the internal reference gene were submitted to Beijing Ruibo Xingke Biotechnology Co., Ltd. for primer synthesis.

[0032] During the flowering period, five female and five male pitcher plants were selected according to their inflorescences. DNA was extracted using the modified CTAB method and stored at -20°C. Figure 1 shown.

[0033] The steps of DNA extraction using the modified CTAB method are as follows:

[0034] (1) Preheat BI solution and 3×CTAB buffer at 65°C. Take a pitcher plant leaf about the size of a fingernail, chop it into small pieces, and place it in a 2 mL centrifuge tube. Add about 6 glass beads and a small amount of polyvinyl pyrrolidone. After sealing, place the centrifuge tube in a container. Pour liquid nitrogen into the container and quickly put it into the grinder when it is almost evaporated.

[0035] (2) Set the grinding machine frequency to 60 Hz for 30 seconds. After grinding, take out the leaf and immediately freeze it in liquid nitrogen or a -20°C freezer. Tap the centrifuge tube and the bottom of the container to prevent the leaf powder from flying away.

[0036] (3) Quickly add 1000 μL of BI solution, vortex mix, let it stand for adsorption, and then place it in a constant temperature oscillator at 65°C for 10 minutes. Invert it upside down for 2 minutes to mix thoroughly.

[0037] (4) Centrifuge at 4°C or room temperature at 12,000 rpm for 10 min, aspirate the supernatant, and discard it;

[0038] (5) Add 1000 μL of 3×CTAB buffer and 8 μL of β-mercaptoethanol, mix thoroughly with a vortex mixer, and place in a 65°C constant temperature oscillator for 2 h, inverting and tilting every 20 min;

[0039] (6) Add approximately 700 μL of chloroform:isoamyl alcohol (24:1) for extraction, mix thoroughly by pouring for 10 min, and centrifuge at 12,000 rpm for 10 min at 4°C or room temperature.

[0040] (7) Take about 900 μL of the supernatant and place it in a 2 mL centrifuge tube. Add about 700 μL of chloroform:isoamyl alcohol (24:1) for a second extraction. Mix well and centrifuge at 4°C or room temperature at 12,000 rpm for 10 min.

[0041] (8) Take 800 μL of supernatant and place it in a 1.5 mL centrifuge tube. Add 700 μL of -20°C pre-cooled isopropanol and pour into the tube to mix. Let it settle for 2 h at -20°C.

[0042] (9) Centrifuge at 4°C or room temperature at 10,000 rpm for 10 min. Discard the supernatant to obtain a white DNA precipitate. Add approximately 500 μL of 75% ethanol (4°C), flick off the white precipitate, wash it clean, and discard the ethanol. Repeat the 75% ethanol wash once. Finally, wash it with 200 μL of anhydrous ethanol (4°C), discard the anhydrous ethanol, and remove any remaining ethanol with a pipette.

[0043] (10) Air-dry the DNA. Place the DNA in a fume hood and air-dry until the white precipitate turns transparent.

[0044] (12) Add 50 μL of TE solution containing 1 μL of RNase (initial concentration 0.1 μg / μL), flick gently to mix, dissolve the DNA precipitate at 37°C, and store the DNA in a -20°C refrigerator.

[0045] The improved CTAB method can improve the concentration and purity of DNA extraction.

[0046] The extracted DNA was used as a template, and PCR amplification was performed using the synthesized NM-MSG primers and the cox1 internal reference gene primers. The PCR reaction system for the NM-MSG male-specific gene and the PCR reaction system for the cox1 internal reference gene amplification were respectively configured.

[0047] PCR reaction system for amplification of the cox1 internal reference gene: The total reaction system is 15 μl, including 7.5 μl of DNA polymerase (2× Taq Master Mix containing Loading Buffer), 1 μl of cox1 forward primer, 1 μl of cox1 reverse primer, 1 μl of DNA template and 4.5 μl of sterile water.

[0048] PCR reaction system for the male-specific gene of NM-MSG: The total reaction system is 15 μl, including 7.5 μl of DNA polymerase (2×Taq MasterMix containing Loading Buffer), 1 μl of NM-MSG forward primer, 1 μl of NM-MSG reverse primer, 1 μl of DNA template and 4.5 μl of sterile water.

[0049] PCR reaction procedure: Place the configured 96-well PCR plate in a PCR instrument (T100 Thermal Cycler), set the temperature conditions, and amplify according to the following program: 95°C, pre-denaturation of DNA template for 5 minutes; 95°C, denaturation for 1 minute; 55°C, annealing for 1 minute; 72°C, extension for 1 minute; repeat the denaturation-annealing-extension procedure 34 times, and then fully extend at 72°C for 5 minutes; the amplified products are stored at 4°C.

[0050] Agarose gel electrophoresis: Use a 1% agarose gel and add 5 μl of cox1 PCR product and 5 μl of NM-MSG PCR product to a single well. Run the gel at 140 V for 30 min. After electrophoresis, observe the amplified bands by UV irradiation.

[0051] Result analysis: Figure 2 As shown in the figure, the cox1 reference gene is a mitochondrial gene shared by both male and female pitcher plants. Under exactly the same conditions, the reference gene primers can amplify the gene sequence in both male and female individuals. Therefore, both female and male plants show the band corresponding to the cox1 reference gene on the amplified band diagram; while the male-specific gene NM-MSG is unique to male plants, so the specific gene primers can only amplify the gene sequence in male individuals, not in females. Therefore, only male pitcher plants show the 523bp band corresponding to the NM-MSG gene on the amplified band diagram, while this band is not shown in female plants.

[0052] The present invention provides a fragment, primer, kit, and method for sex identification of Nepenthes. A fragment capable of identifying the sex of Nepenthes is developed and screened, and primers for designing specific molecular markers are used from the fragment. The primers, including a forward primer and a reaction primer, amplify DNA extracted from Nepenthes, generating distinct bands that are organized into a fingerprint pattern, resulting in a stable and distinct amplification result, allowing Nepenthes of different sexes to be distinguished. This method exhibits excellent stability and is applicable to different Nepenthes individuals.

[0053] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, ordinary technicians in this field can make various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

1. A primer for detecting specific molecular markers for sex identification of Nepenthes, characterized in that: The primers are designed based on the nucleotide sequence shown in SEQ ID NO.1, and include a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

2. Use of the primer according to claim 1 in sex identification of Nepenthes.

3. A kit for sex identification of Nepenthes, characterized in that: The kit includes the primers described in claim 1.

4. A method for sex identification of pitcher plants, characterized in that: The following steps are involved: 1) Extract DNA from the pitcher plant to be identified; 2) using DNA of the pitcher plant to be identified as a template and performing PCR amplification using the primers described in claim 1 or the kit described in claim 3; 3) Detect the PCR amplification product. If a 523 bp band is detected, the pitcher plant to be identified is a male pitcher plant. When the amplified product does not show a 523 bp band, the pitcher plant to be identified is a female pitcher plant.

5. The method according to claim 4, characterized in that In step 2), the reaction system for PCR amplification is: a total reaction system of 15 μL includes 7.5 μL of DNA polymerase, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of DNA of the pitcher plant to be identified, and 4.5 μL of sterile water, wherein the nucleotide sequences of the forward primer and the reverse primer are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

6. The method according to claim 4 or 5, characterized in that The PCR reaction procedure is as follows: pre-denaturation of the DNA of the pitcher plant to be identified at 95°C for 5 minutes; denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and extension at 72°C for 1 minute. The denaturation-annealing-extension procedure is repeated 34 times, followed by full extension at 72°C for 5 minutes.

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