Fragment, primer, kit and method for identifying sex of nepenthes mirabilis

Abstract

The invention discloses a segment, a primer, a kit and a method for sex identification of nepenthes, a sex linkage region of a Y chromosome of the nepenthes is locked by utilizing whole genome association analysis, a male specific segment is further screened, the primer of a specific molecular marker is designed by utilizing the segment, the primer comprises a forward primer and a reaction primer, and the reaction primer comprises a forward primer and a reverse primer. According to the method, the extracted DNA of the nepenthes is subjected to PCR amplification to obtain an amplification product, and the amplification product is subjected to agarose gel electrophoresis, so that different bands are generated and arranged into a fingerprint spectrum, a stable and obvious amplification result is obtained, and nepenthes with different genders can be distinguished. The method is used for identifying the early sex of the nepenthes, is excellent in stability, is suitable for different individuals of the nepenthes, and is of great significance to sex identification of the nepenthes.

Description

Technical Field

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

[0002] Nepenthes mirabilis is a climbing herb with lanceolate leaves. Tendrils 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 is highly scientific and ornamental. Nepenthes flowers are unisexual and dioecious. When the male flowers bloom, they emit a special smell to attract insects for pollination. Nepenthes reproduces by 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 by creeping branches on the ground and taking root. At present, the identification of the sex of Nepenthes mainly depends on the differentiation of the morphology of male and female flowers of the reproductive organs, such as Figure 1 As shown, sex identification is not possible in the early stage of young plants before the differentiation of male and female flowers. Therefore, the inability to identify sex in the early stage has brought great obstacles to field surveys and sample collection of Nepenthes, as well as the horticultural breeding and cultivation of this species and artificial hybridization.

[0003] In previous research reports, Scharmann et al. (2019) performed double restriction site sequencing (ddRAD-seq) on three Nepenthes species and found the male-specific site of Nepenthes. Then, the transcriptome of the male inflorescence of Indian Nepenthes (N.khasiana) was sequenced and assembled, and the male-specific gene (DYT1) was obtained. The corresponding molecular markers were developed, which are F: 5'-AATTCACTGATTCGGATCACG-3; R: 5'-CGATCGCGTCGCAAAGTATG-3'. At the same time, the mitochondrial gene cox1 shared by female and male Nepenthes plants (primers are F: 5'-GGAGGAGTTGATTTAGC-3'; R: 5'-AAGGCTGGAGGGCTTTGTAC-3') was used as an internal reference gene. Based on PCR experiments, the specificity of the DYT1 gene in male Nepenthes plants was verified. However, this study has certain limitations. For example, the double restriction site sequencing method has a low genome coverage rate, and the DYT1 gene information obtained may be incomplete. At the same time, the PCR product length of the primers developed based on the DYT1 gene is relatively small, resulting in unclear agarose gel electrophoresis results. 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 needs further verification. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a fragment, primer, kit and method for sex identification of Nepenthes.

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

[0006] Use of the fragment described in the first aspect of the present invention in identifying the sex of Nepenthes.

[0007] 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.

[0008] 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.

[0009] The invention relates to the use of the primers described in the second aspect of the invention in sex identification of Nepenthes.

[0010] The third aspect of the present invention provides a kit for identifying the sex of Nepenthes, wherein the kit comprises the primers described in the second aspect of the present invention.

[0011] A fourth aspect of the present invention provides a method for identifying sex of a pitcher plant, comprising the following steps:

[0012] 1) Extracting DNA from the pitcher plant to be identified;

[0013] 2) using the 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;

[0014] 3) Detect PCR amplification products. When the amplification products show:

[0015] When the band is 500 bp, the pitcher plant to be identified is a male pitcher plant;

[0016] Or, when the amplified product does not show a 500 bp band, the pitcher plant to be identified is a female pitcher plant.

[0017] Preferably, in the step 2), the reaction system for PCR amplification is: the total reaction system is 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 in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0018] 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.

[0019] Compared with the prior art, 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, develops primers, kits and methods for molecular markers for the male-specific fragments of Nepenthes, and optimizes molecular experiments for identifying the early sex of Nepenthes. The identification method has excellent stability, is applicable to different individuals of Nepenthes, and is of great significance for the sex identification of Nepenthes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a characteristic diagram of male and female inflorescences of Nepenthes;

[0021] Figure 2 This is the PCR amplification band diagram. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0023] Example

[0024] To obtain specific gene sequences, based on the third-generation sequencing of female and male pitcher plants, we have a complete reference genome of male and female plants. We deeply resequenced 80 different pitcher plant individuals and determined the Y chromosome linkage region of pitcher plants through genome-wide association analysis (GWAS). We further sequenced the transcriptomes of male and female flower organs of Nepenthes, performed expression difference analysis, enrichment analysis, etc., compared and analyzed the molecular differences between male and female individuals, and mined out male-specific fragments of Nepenthes, recorded as NM-MSG. Through bioinformatics methods and data comparison, we determined that the NM-MSG sequence was different from the previous DYT1 gene. And the female pitcher plants do not contain the above fragments, so primers can be designed based on this fragment to further identify the sex of Nepenthes.

[0025] The nucleotide sequence of NM-MSG is shown in SEQ ID NO.1:

[0026]

[0027] Design primers. The specific fragment is unique to male Nepenthes, so primers are designed based on this fragment. After obtaining the sequence of the specific fragment NM-MSG, primers are designed in primer3plus (https: / / www.primer3plus.com), and the specificity of the primers is preliminarily verified in NCBIblast. The primers can amplify the male-specific fragment by PCR. The primers designed to obtain NM-MSG include forward primers and reverse primers;

[0028] The nucleotide sequence of the forward primer is shown in SEQ ID NO. 2: 5'-TTCTGCCCAGCTCAATGGAG-3';

[0029] The nucleotide sequence of the reverse primer is shown in SEQ ID NO. 3: 5'-CCGGAAGACAGCATCACAGT-3'.

[0030] The mitochondrial gene cox1 shared by female and male pitcher plants was used as the internal reference gene. Under exactly the same conditions, the primers of the internal reference gene can amplify the gene sequence in both male and female individuals, while the primers of NM-MSG can only amplify the gene sequence in male individuals but not in females.

[0031] The primers for the internal reference gene cox1 are: forward primer (F): 5'-GGAGGAGTTGATTTAGC-3';

[0032] Reverse primer (R): 5'-AAGGCTGGAGGGCTTTGTAC-3'.

[0033] 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.

[0034] During the flowering period, five female and five male plants of Nepenthes were selected according to their inflorescences. DNA was extracted using the modified CTAB method and stored at -20°C. The inflorescences of the female and male plants were as follows: Figure 1 shown.

[0035] The steps of the modified CTAB method for DNA extraction are as follows:

[0036] (1) Preheat BI solution and 3× CTAB buffer at 65°C, take a fingernail-sized pitcher plant leaf, cut it into pieces and mash it, place it in a 2 mL centrifuge tube, add about 6 glass beads and a small amount of polyvinyl pyrrolidone, seal the tube, place the centrifuge tube in a container, pour liquid nitrogen into the container, and quickly put it into a grinder when it is almost evaporated;

[0037] (2) Set the grinding machine frequency to 60 Hz for 30 seconds. After grinding, take out the leaves and immediately freeze them 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.

[0038] (3) Quickly add 1000 μL of BI solution, vortex mix, let stand for adsorption, place in a 65°C constant temperature oscillator for 10 min, and mix by inverting for 2 min;

[0039] (4) Centrifuge at 4°C or room temperature at 12,000 rpm for 10 min, aspirate the supernatant and discard it;

[0040] (5) Add 1000 μL 3× CTAB buffer and 8 μL β-mercaptoethanol, mix thoroughly with a vortex mixer, and place on a 65°C constant temperature oscillator for 2 h, inverting and tilting every 20 min;

[0041] (6) Add about 700 μL of chloroform:isoamyl alcohol (24:1) for extraction, pour and mix for 10 min, and centrifuge at 4°C or room temperature at 12,000 rpm for 10 min;

[0042] (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) to extract for the second time. Mix well and centrifuge at 4°C or room temperature at 12,000 rpm for 10 min.

[0043] (8) Take 800 μL of supernatant in a 1.5 mL centrifuge tube, add 700 μL of -20°C precooled isopropanol, pour and mix, and precipitate for 2 h at -20°C;

[0044] (9) Use a 4℃ or room temperature centrifuge at 10000rpm for 10min. Discard the supernatant to obtain a white DNA precipitate; add about 500μL of 75% ethanol (4℃), flick off the white precipitate, wash it clean and pour out the ethanol. Repeat the 75% ethanol wash once; finally, wash it with 200μL of anhydrous ethanol (4℃), pour out the anhydrous ethanol, and use a pipette to remove the remaining ethanol;

[0045] (10) Air-dry the DNA. Place the DNA in a fume hood and air-dry until the white precipitate turns transparent.

[0046] (12) Add 50 μL of TE solution containing 1 μL of RNase (initial concentration 0.1 μg / μL), flick to mix, dissolve the DNA precipitate at 37°C, and store the DNA in a -20°C refrigerator.

[0047] The concentration and purity of DNA extraction can be improved by the improved CTAB method.

[0048] The extracted DNA was used as a template, and PCR amplification was performed using the synthesized NM-MSG primers and cox1 internal reference gene primers, respectively, to prepare a PCR reaction system for NM-MSG male-specific gene and a PCR reaction system for cox1 internal reference gene amplification, wherein

[0049] 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.

[0050] PCR reaction system of NM-MSG male-specific 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 NM-MSG forward primer, 1 μl of NM-MSG reverse primer, 1 μl of DNA template and 4.5 μl of sterile water.

[0051] 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 procedure: 95°C, DNA template pre-denaturation for 5 minutes; 95°C, denaturation for 1 minute; 55°C, annealing for 1 minute; 72°C, extension for 1 minute; after repeating the denaturation-annealing-extension procedure 34 times, fully extend at 72°C for 5 minutes; the amplified product is stored at 4°C.

[0052] Agarose gel electrophoresis: Use 1% agarose gel, add 5 μl of cox1 PCR product and 5 μl of NM-MSG PCR product to a single well at the same time, 140 volts, electrophoresis time 30 minutes. After the electrophoresis, observe the amplified bands by UV irradiation.

[0053] Result analysis: Figure 2 As shown, the cox1 internal reference gene is a mitochondrial gene shared by female and male pitcher plants. Under exactly the same conditions, the internal reference gene primers can amplify the gene sequence in both male and female individuals. Therefore, both female and male plants on the amplified band diagram show the band corresponding to the cox1 internal reference gene; while the male plant's specific gene NM-MSG is unique to the male plant, so the specific gene primers can only amplify the gene sequence in male individuals, not in females. Therefore, on the amplified band diagram, only male pitcher plants show the 500bp band corresponding to the NM-MSG gene, while this band is not shown in female plants.

[0054] The present invention provides a fragment, primer, kit and method for identifying the sex of Nepenthes, wherein a fragment capable of identifying the sex of Nepenthes is developed and screened, and primers for specific molecular markers are designed using the fragment, wherein the primers, including forward primers and reaction primers, amplify the DNA extracted from Nepenthes, generate different bands and arrange them into fingerprints, obtain a stable and obvious amplification result, and Nepenthes of different sexes can be distinguished. The method has excellent stability and is suitable for different individuals of Nepenthes.

[0055] 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, and such changes all fall within the scope of protection of the present invention.

Claims

1. A fragment for identifying the sex of a pitcher plant, characterized in that: The nucleotide sequence of the fragment is shown in SEQ ID NO.

1.

2. Use of the fragment according to claim 1 in identifying the sex of Nepenthes.

3. A primer for identifying sex-specific molecular markers of Nepenthes, characterized in that: The primers are designed based on the nucleotide sequence of the fragment described in SEQ ID NO.

1.

4. The primer according to claim 3, characterized in that 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.

5. Use of the primer according to claim 3 or 4 in sex identification of Nepenthes.

6. A kit for identifying the sex of Nepenthes, characterized in that: The kit comprises the primers described in claim 3 or 4.

7. A method for identifying the sex of a pitcher plant, characterized in that: The following steps are involved: 1) Extracting DNA from the pitcher plant to be identified; 2) using the 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 6; 3) Detect PCR amplification products. When the amplification products show: When the band is 500 bp, the pitcher plant to be identified is a male pitcher plant; Or, when the amplified product does not show a 500 bp band, the pitcher plant to be identified is a female pitcher plant.

8. The method according to claim 7, characterized in that In the step 2), the reaction system for PCR amplification is: the total reaction system is 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 in SEQ ID NO.2 and SEQ ID NO.3, respectively.

9. The method according to claim 7 or 8, characterized in that: 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.

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