Primers and probes for distinguishing field-grown wild watermelon seedlings and application thereof

Abstract

The application provides a primer and a probe for distinguishing field wild watermelon seedlings and application thereof, and belongs to the technical field of molecular agricultural biotechnology and plant molecular identification technology. The primer is YXG-95K-F and YXG-95K-R. The nucleotide sequence of the probe is [5'FAM]-GATGGATATATATCGTATAATTCTTATCGTAT(THF)ATTCTTATATATATT-[3'C3spacer]. The primer and the probe are used for distinguishing or identifying whether it is wild watermelon. The application can detect watermelon and wild watermelon leaf tissues by using molecular biology methods such as RPA, and molecular identification can be realized by whether a fluorescent signal exists, so that the watermelon and the wild watermelon can be reliably and effectively distinguished.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and plant molecular identification technology, and relates to primers and probes for distinguishing wild watermelon seedlings in the field and their applications. In particular, it relates to a molecular marker, specific primer pair, fluorescent probe, and their applications based on chloroplast genome or nuclear genome specific sites for distinguishing cultivated watermelon (Citrullus lanatus) of the Cucurbitaceae family and wild watermelon seedlings (Hibiscus trionum) of the Malvaceae family. Background Technology

[0002] Watermelon cultivation is an important economic crop in my country. Wild watermelon seedlings (Hibiscus trionum) are a common field weed. In their seedling stage, they are morphologically similar to watermelon seedlings, making them difficult to distinguish. This often leads to the accidental removal of watermelon seedlings or the omission of weeds during manual or mechanical weeding, resulting in economic losses or the spread of weed infestation. Traditional morphological identification methods are highly dependent on experience and inefficient, failing to meet the needs of modern precision agriculture.

[0003] Developing a rapid, accurate molecular detection method capable of identification at the seedling stage or even the seed stage is of great significance for the safe production, field management, and germplasm purity identification of watermelons. Since watermelon and wild watermelon seedlings belong to different families, are distantly related, and have significant genomic differences, this provides a good foundation for developing specific molecular markers. Summary of the Invention

[0004] To address the aforementioned issues, this invention screens specific molecular markers based on chloroplast or nuclear genome-specific sites, and designs DNA molecular probes that can be used to distinguish between watermelon and wild watermelon. This provides primers and probes that can quickly and accurately distinguish between watermelon and wild watermelon, along with their applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A primer and probe for distinguishing wild watermelon seedlings in the field, wherein the primers are YXG-95K-F and YXG-95K-R; The nucleotide sequence of the primer YXG-95K-F is shown in SEQ ID NO: 1; The nucleotide sequence of the primer YXG-95K-R is shown in SEQ ID NO: 2; The nucleotide sequence of the probe is [5'FAM]-GATGGATATATATCGTATAATTCTTATCGTAT(THF)ATTCTTATATATATAT-[3'C3spacer].

[0006] An application of the above-mentioned primers and probes, wherein the primers and probes are used to distinguish or identify watermelon from wild watermelon.

[0007] A kit for distinguishing wild watermelon seedlings in the field, the kit comprising the aforementioned primers and probes.

[0008] Furthermore, the kit includes the primer YXG-95K-F, the primer YXG-95K-R, the probe, reacting buffer, E mix, and P mix.

[0009] Furthermore, the kit also includes nucleic acid test strips.

[0010] A method for distinguishing wild watermelon seedlings in the field, wherein the method uses the above-mentioned primers and probes to amplify the DNA of the sample to be tested in order to distinguish or identify watermelon from wild watermelon; Alternatively, the method involves using the aforementioned kit to amplify the DNA of the sample to be tested in order to distinguish or identify watermelons from wild watermelons.

[0011] Furthermore, the method includes the following specific steps: DNA is extracted from the sample to be tested to obtain template DNA; The template DNA was amplified using the primers and probes described above to obtain the amplification product. The amplification product was tested using a nucleic acid test strip to determine whether the sample was watermelon or wild watermelon. When both the control line (C) and the test line (T) show bands, the sample to be tested is a wild watermelon; When only the control line (C) shows a band but the test line (T) shows no band, the sample to be tested is a watermelon.

[0012] Furthermore, the method includes the following specific steps: DNA is extracted from the sample to be tested to obtain template DNA; The template DNA was amplified using the kit described above to obtain the amplification product. The amplification product was tested using a nucleic acid test strip to determine whether the sample was watermelon or wild watermelon. When both the control line (C) and the test line (T) show bands, the sample to be tested is a wild watermelon; When only the control line (C) shows a band but the test line (T) shows no band, the sample to be tested is a watermelon.

[0013] Furthermore, the amplification system includes: primer YXG-95K-F, primer YXG-95K-R, probe, DNA of the sample to be tested, reacting buffer, E mix, P mix, and water.

[0014] Furthermore, the amplification temperature was 40℃ and the time was 30 min.

[0015] The beneficial effects of the primers and probes of the present invention for distinguishing wild watermelon seedlings in the field and their applications are as follows: Based on the alignment results of wild watermelon seedlings and watermelon chloroplast genomes, several differential sites were identified, including a significant differential site in the 95kbp region. This single site can distinguish wild watermelon seedlings from watermelons, making it an ideal site for molecular markers. Sequences approximately 400bp upstream and downstream of this differential site were extracted as references, and primers were designed based on these homologous sequences. PCR amplification using DNA from both wild watermelon seedlings and watermelons as templates yielded single, bright bands of the expected size, demonstrating the existence of the differential site fragment obtained from chloroplast genome sequence analysis and its suitability for amplification and subsequent identification. The PCR amplification products were subjected to Sanger sequencing, and the sequencing results were compared. The comparison results showed that there was a 29bp differential site at this site that could distinguish wild watermelon seedlings from watermelons. Based on this site, an oligonucleotide fluorescent probe was designed, which can be used to detect wild watermelon seedlings and watermelon leaf tissues using molecular biology methods such as RPA (Recombinase Polymerase Amplification). Molecular identification can be achieved by the presence or absence of fluorescent signals, thus reliably and effectively distinguishing wild watermelon seedlings from watermelons. This invention develops a primer and probe for distinguishing wild watermelon seedlings in the field. Using this primer and probe, watermelons and wild watermelons can be accurately and efficiently distinguished, which greatly improves the identification and management efficiency of wild watermelons, effectively controls the damage caused by wild watermelons (weeds), and has extremely high economic value. Attached Figure Description

[0016] Figure 1 This is the comparison result of chloroplasts of wild watermelon and watermelon in Example 1 of the present invention; wherein, OL_628869 is wild watermelon and NC_032008 is watermelon, and the gene sequence difference site between the two is the position marked at 95760 bp in the figure; Figure 2 The images are the PCR amplification electrophoresis bands of wild watermelon seedlings and watermelon molecular markers in Example 1 of this invention; where M represents Marker, YXG represents wild watermelon, and XG represents watermelon, and the band size is approximately 400bp. Figure 3 This is a comparison of sequencing results of the differential loci (95K) between wild watermelon seedlings and watermelons in Example 1 of the present invention, wherein, from top to bottom, they are wild watermelons and watermelons; Figure 4This refers to the detection results of the single nucleic acid test strip in Example 2 of the present invention on the DNA amplification products of watermelon and wild watermelon; wherein, a positive result indicates that the kit contains a positive reagent, and a negative result indicates that the kit contains a negative reagent. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it.

[0018] Experimental apparatus: High-speed refrigerated centrifuge: ThermoFisher, Micro 21R; Constant temperature water bath: Jinghong, DK-S24; Ultra-micro spectrophotometer: ThermoFisher, NanoDrop2000; PCR instrument: ThermoFisher, Applied Biosystems VeritiPro; Gel electrophoresis system: Liuyi, DYY-8C; Gel imaging system: VILBER, FUSION FX7; Experimental reagents: Plant genomic DNA extraction kit: Tiangen, DP305; 2 × Phanta Max Master Mix (DyePlus): Novizan, P525; D2000 molecular weight standard: Tiangen, MD114.

[0019] Example 1: Design of a molecular marker probe for distinguishing between watermelon and wild watermelon 1) Sample grinding: Liquid nitrogen was used to grind the leaf tissues of watermelon plants and wild watermelon plants into uniform powder.

[0020] 2) DNA extraction: Following the instructions of the plant genomic DNA extraction kit, DNA was extracted from the leaf tissues of watermelon and wild watermelon plants using the kit as template DNA, and the concentration of the corresponding DNA was detected.

[0021] 3) Chloroplast genome acquisition: Based on existing data in public databases and samples stored in our laboratory, we performed whole-genome sequencing of chloroplasts. After removing low-quality data, we assembled the clean reads to obtain relatively complete chloroplast genome data.

[0022] The chloroplast genomes of watermelon and wild watermelon were retrieved from GenBank, and the search numbers are shown in Table 1.

[0023] Table 1. Chloroplast genome sequences of the study samples (Genbank search numbers)

[0024] 4) Sequence Alignment: The chloroplast genomes of watermelon and wild watermelon were aligned using MAFFT software. By comparing and analyzing the chloroplast genome sequences of watermelon and wild watermelon, specific sites showing significant differences between the two plants could be identified, such as… Figure 1 As shown.

[0025] 5) PCR primer design: Based on the alignment results of the chloroplast genomes of watermelon and wild watermelon, differential sites that can distinguish watermelon from wild watermelon were selected. The differential sites and their upstream and downstream fragments of approximately 270 bp and approximately downstream fragments of approximately 132 bp were extracted to design primers (wherein, the differential sites of watermelon and their upstream and downstream sequences are shown in SEQ ID NO: 3, and the differential sites of wild watermelon and their upstream and downstream sequences are shown in SEQ ID NO: 4). The PCR forward primer YXG-95K-F (5'-atatcaaacggacctccccagaaggt-3', i.e., as shown in SEQ ID NO: 1) and the reverse primer YXG-95K-R (5'-ctacatgaagaacataagccaga-3', i.e., as shown in SEQ ID NO: 2) were obtained and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0026] 6) PCR amplification verification: Configure the PCR reaction system according to Table 2, set the PCR program according to Table 3, and perform PCR amplification on the template DNA of watermelon and wild watermelon to obtain the corresponding PCR products.

[0027] Table 2 PCR system

[0028] Table 3 PCR Procedure

[0029] 7) Gel electrophoresis: The band size of each PCR product was detected using 1.5% agarose gel electrophoresis. The results are as follows: Figure 2 As shown in the figure, the results indicate that PCR amplification of approximately 400 bp upstream and downstream of the differential site was performed, and the agarose gel electrophoresis results were as expected, indicating that the target fragment was amplified in both watermelon and wild watermelon.

[0030] 8) Sequencing and Sequence Alignment: PCR products with electrophoretic band sizes matching the expected results were sent to Youkang Biotechnology for sequencing. DNAMAN was used to align differentially expressed sites in the chloroplast genomes of watermelon and wild watermelon. After sequencing the amplified fragments from watermelon and wild watermelon at Youkang Biotechnology, DNAMAN analysis of the sequencing results allowed for the screening of differentially expressed sites, such as… Figure 3 As shown.

[0031] 9) Probe design: Following the probe design principle, an oligonucleotide probe with a length of 45-50nt was designed at a specific site on the watermelon chloroplast genome. A FAM fluorescent group was labeled at the 5' end, and a C3-spacer was modified at the 3' end. A d-spacer (THF tetrahydrofuran) was inserted in the middle of the probe at a distance of more than or equal to 35nt from the 5' end.

[0032] Based on the sequence at the 95kbp differential site, fluorescent molecularly labeled oligonucleotide probes were designed, and the results are shown in Table 4.

[0033] Table 4. Fluorescent probe sequences based on specific sites

[0034] Example 2: Establishment of the detection kit and verification experiment for detecting watermelon and wild watermelon. 1) Kit: The kit for distinguishing between watermelon and wild watermelon includes: primers YXG-95K-F / YXG-95K-R, probe (YXG-95K), reacting buffer, E mix, P mix, water and single nucleic acid test strip (AMP Future, WLFS8206). The nucleotide sequence of the probe (YXG-95K) is [5'FAM]-GATGGATATATATCGTATAATTCTTATCGTAT(THF)ATTCTTATATATATT-[3'C3spacer].

[0035] Specifically, a FAM fluorescent group is labeled at the 5' end of the probe, and a C3-spacer is used to modify the 3' end. A d-spacer (THF tetrahydrofuran) is inserted at a distance of 35 nt from the 5' end in the middle of the probe. The single nucleic acid test strip has a control line (C line) in the quality control area and a test line (T line) in the detection area; one end of the nucleic acid test strip is equipped with a sample application pad.

[0036] 2) Verification Experiment 21) DNA extraction: Following the instructions of the plant genomic DNA extraction kit, DNA was extracted from the leaf tissues of watermelon and wild watermelon plants using the kit as template DNA, and the concentration of the corresponding DNA was detected.

[0037] 22) DNA Amplification: Using a kit including primers YXG-95K-F / YXG-95K-R and probe (YXG-95K), prepare the amplification system according to the system in Table 5, using sterile water instead of DNA template as a negative control. After preparing the amplification system, add 2 μL of starter and incubate in a 40℃ water bath for 30 min to obtain the amplification product for later use.

[0038] Table 5. Sample DNA isothermal amplification system

[0039] 23) Single nucleic acid test strip detection: Take 10 μL of amplification product, dilute it 10-fold with Diluent buffer, and add it to the single nucleic acid test strip. After 5 minutes of chromatography reaction, check the test line (T) and control line (C). The results are as follows: Figure 4 As shown.

[0040] 24) Experimental results: Figure 4 The results of the single-strand nucleic acid test strip showed that bands appeared at the control line (C), indicating that the detection was effective. In this invention, the oligonucleotide fluorescent probe successfully bound to the DNA of wild watermelon leaves, causing the fluorophore to develop color and a band to appear at the detection line (T). However, the watermelon leaf DNA did not bind to the probe, resulting in no color development or band at the detection line (T). Based on the detection results, this molecular marker probe can be used to distinguish between watermelon and wild watermelon.

[0041] In practical applications, DNA is directly extracted from the sample to be tested, and after DNA amplification according to the above method, the amplified product is detected using a nucleic acid test strip according to the above method, which can determine whether the sample to be tested is watermelon or wild watermelon.

[0042] 3) Summary: Based on the alignment results of wild watermelon seedlings and watermelon chloroplast genomes, several differentially expressed sites were identified, including a significant differential site within a 95kbp region. This single site is ideal for distinguishing wild watermelon seedlings from watermelons and serves as a molecular marker. Approximately 400bp upstream and downstream sequences from this differential site were extracted as references, and primers were designed based on these homologous sequences. PCR amplification using DNA from both wild watermelon seedlings and watermelons as templates yielded single, bright bands of the expected size, demonstrating the existence of the differentially expressed site fragment identified in the chloroplast genome sequence analysis and its suitability for amplification and subsequent identification.

[0043] Sanger sequencing was performed on the PCR amplification products, and the sequencing results were compared. The comparison results showed that there was a 29bp differential site at this locus that could separate wild watermelon seedlings from watermelons. Based on this site, an oligonucleotide fluorescent probe was designed, which can be used to detect wild watermelon seedlings and watermelon leaf tissues using molecular biology methods such as RPA (Recombinase Polymerase Amplification). Molecular identification can be achieved by the presence or absence of fluorescent signals, thus reliably and effectively distinguishing wild watermelon seedlings from watermelons.

[0044] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. Those skilled in the art can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer and probe for distinguishing wild watermelon seedlings in the field, characterized in that, The primers are YXG-95K-F and YXG-95K-R; The nucleotide sequence of the primer YXG-95K-F is shown in SEQ ID NO: 1; The nucleotide sequence of the primer YXG-95K-R is shown in SEQ ID NO: 2; The nucleotide sequence of the probe is [5'FAM]-GATGGATATATATCGTATAATTCTTATCGTAT(THF)ATTCTTATATATATAT-[3'C3spacer].

2. An application of the primer and probe according to claim 1, characterized in that, The primers and probes are used to distinguish or identify wild watermelons.

3. A reagent kit for distinguishing wild watermelon seedlings in the field, characterized in that, The kit includes the primers and probes as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit includes primer YXG-95K-F, primer YXG-95K-R, probe, reacting buffer, E mix, and P mix.

5. The kit according to claim 3 or 4, characterized in that, The kit also includes nucleic acid test strips.

6. A method for distinguishing wild watermelon seedlings in a field, characterized in that, The method uses the primers and probes described in claim 1 to amplify the DNA of the sample to be tested, in order to distinguish or identify whether it is a wild watermelon; Alternatively, the method may involve amplifying the DNA of the sample to be tested using the kit described in any one of claims 3-5, in order to distinguish or identify whether it is a wild watermelon.

7. The method for distinguishing wild watermelon seedlings in the field according to claim 6, characterized in that, The method includes the following specific steps: DNA is extracted from the sample to be tested to obtain template DNA; The template DNA was amplified using the primers and probes described in claim 1 to obtain the amplification product. The amplification product was tested using a nucleic acid test strip to determine whether the sample was a wild watermelon. When both the control line (C) and the test line (T) show bands, the sample to be tested is a wild watermelon.

8. The method for distinguishing wild watermelon seedlings in the field according to claim 6, characterized in that, The method includes the following specific steps: DNA is extracted from the sample to be tested to obtain template DNA; The template DNA was amplified using the kit described in any one of claims 3-5 to obtain the amplification product; The amplification product was tested using a nucleic acid test strip to determine whether the sample was a wild watermelon. When both the control line (C) and the test line (T) show bands, the sample to be tested is a wild watermelon.

9. The method for distinguishing wild watermelon seedlings in the field according to any one of claims 6-8, characterized in that, The amplification system includes: primer YXG-95K-F, primer YXG-95K-R, probe, DNA of the sample to be tested, reacting buffer, E mix, P mix, and water.

10. The method for distinguishing wild watermelon seedlings in the field according to any one of claims 6-8, characterized in that, The amplification temperature was 40℃ and the time was 30 min.

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