Visual poplar sex detection primer based on RPA-LFD and RPA-CRISPR / Cas 12a, probe and crRNA and application thereof

Abstract

The invention discloses a poplar sex visual detection primer, a probe and crRNA based on RPA-LFD and RPA-CRISPR / Cas 12a and application of the poplar sex visual detection primer, the probe and the crRNA, and belongs to the technical field of gene detection. Specific primers (SEQ ID NO.1 / 2 or SEQ ID NO.5 / 6) are designed on the basis of a poplar MSL sex determination gene, two visual detection modules, namely a lateral flow chromatography test strip module or a CRISPR / Cas 12a signal output module, are combined to form an RPA-LFD detection path combined with an NFO probe and an RPA-CRISPR / Cas 12a detection path based on Cas 12a signal amplification, and sex determination can be rapidly completed under the condition of constant temperature. The two systems can be read by naked eyes through LFD test strips, the detection operation is simple and convenient, the time is short, and the method is suitable for rapid sex identification of the poplar under laboratory and field conditions.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and in particular relates to a primer, probe and crRNA for visual detection of sex in poplar trees based on RPA-LFD and RPA-CRISPR / Cas12a, and their applications. Background Technology

[0002] Poplar, belonging to the genus *Populus* of the family Salicaceae, is one of the world's most important fast-growing timber species. It is widely used in the production of engineered wood products, papermaking, furniture, and urban greening, making it a crucial industrial raw material species in my country and globally, with significant social and economic value. Simultaneously, poplar possesses strong carbon sequestration and ecological restoration capabilities, and is widely used for windbreak and sand fixation, soil and water conservation, and other purposes, demonstrating significant value in improving the ecological environment and promoting the carbon cycle. Among them, the black poplar variety has become an important species in the construction of fast-growing and high-yield forests and ecological environment management. It is widely used in my country's timber forests and shelterbelt construction, playing a vital role in promoting the optimization and sustainable development of the forestry industry structure, and has significant application value in forestry production, the construction of fast-growing and high-yield forests, and ecological restoration.

[0003] Poplar trees are dioecious plants, with female trees releasing large amounts of seed fluff (also known as "flying catkins") when they mature in spring. The seeds have a thick layer of white downy hairs that disperse in the air. This fluff, along with the pollen and dust it carries, can trigger allergic reactions, asthma, or respiratory diseases, negatively impacting residents' health. Furthermore, the flying catkins, when concentrated in high concentrations, are flammable, posing a potential fire risk. The production of flying catkins primarily originates from the seed dispersal structure of female trees; therefore, one effective way to control flying catkins at their source is to accurately identify the sex of poplar trees during the breeding and afforestation stages, prioritizing the selection of male trees or controlling the sex ratio. Establishing a rapid, simple, and visual method for poplar sex identification is of great significance for reducing urban flying catkin pollution and guiding tree breeding and landscape design.

[0004] Traditional methods for sex identification in poplar trees mainly include morphological observation and molecular biological identification. Morphological observation requires identifying sex through floral organs after the plant reaches flowering stage, which is time-consuming and highly susceptible to environmental influences. Molecular detection methods such as PCR and qPCR can achieve identification in the seedling stage, but they usually rely on expensive experimental equipment, complex operating procedures, and strict experimental conditions, making them unsuitable for rapid field detection. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technology developed in recent years, with advantages such as low reaction temperature, fast speed, and no need for sophisticated instruments, making it particularly suitable for rapid on-site detection in resource-constrained environments. To meet the needs of field samples or on-site detection, an NFO probe can be introduced into the RPA reaction and combined with a lateral flow dipstick (LFD) to visualize the amplification results by observing the color changes of the control and test lines on the test strip. In addition, RPA amplification can also be coupled with the CRISPR / Cas 12a system and used with test strips for visual detection. This method leverages the target sequence-dependent activation and cleavage characteristics of the CRISPR / Cas 12a system: guided by crRNA, Cas 12a recognizes the target sequence and activates its non-specific cleavage activity, cleaving single-stranded DNA reporter molecules to achieve highly sensitive detection of the target nucleic acid. However, there are currently no publicly reported RPA-LFD detection systems for poplar sex, nor are there any visual detection methods for poplar sex based on RPA-CRISPR / Cas 12a. Rapid and visual detection of poplar sex has significant application value, applicable to early sex determination, fluff control, and breeding material selection. Therefore, this paper provides primers, probes, and crRNA for visual detection of poplar sex based on RPA-LFD and RPA-CRISPR / Cas 12a, along with their applications, meeting the urgent need in this field for efficient, simple, and rapid detection technologies. Summary of the Invention

[0005] In view of the above-mentioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a poplar sex visualization detection primer, probe and crRNA based on RPA-LFD and RPA-CRISPR / Cas 12a. Another technical problem to be solved by the present invention is to provide an application of the poplar sex visualization detection primer, probe and crRNA based on RPA-LFD and RPA-CRISPR / Cas 12a in poplar sex identification.

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] A recombinase polymerase amplification detection system for sex identification of poplar trees, the system being used for sex identification of American black poplar (Populus deltoides) and / or European and American poplar (Populus × euramericana), and comprising:

[0008] (a) A first primer pair consisting of a nucleotide sequence as shown in SEQ ID NO.1 and a nucleotide sequence as shown in SEQ ID NO.2; and / or a second primer pair consisting of a nucleotide sequence as shown in SEQ ID NO.5 and a nucleotide sequence as shown in SEQ ID NO.6;

[0009] (b) A detection module used in conjunction with the primer pair in (a), wherein the detection module is selected from any of the following:

[0010] (i) Sideflow chromatography test strip detection module; or

[0011] (ii) CRISPR / Cas 12a signal output detection module.

[0012] In some embodiments, when the detection module is a lateral flow chromatography test strip detection module, the 5' end of the nucleotide sequence shown in SEQ ID NO.1 and / or SEQ ID NO.5 is covalently linked to a first reporter molecule, and the system further includes a probe corresponding to the primer pair, the 5' end of which is covalently linked to a second reporter molecule different from the first reporter molecule.

[0013] In some embodiments, the first reporter molecule is biotin, the second reporter molecule is fluorescein (FITC), and the detection line of the lateral flow chromatography test strip is coated with an antibody against the second reporter molecule.

[0014] In some embodiments, the nucleotide sequence of the probe is as shown in SEQ ID NO.3 or SEQ ID NO.7, and it contains tetrahydrofuran (THF) residues and an extension blocking group at the 3' end.

[0015] In some embodiments, when the detection module is a CRISPR / Cas 12a signal output detection module, the system further includes crRNA corresponding to the primer pair, wherein the nucleotide sequence of the crRNA corresponding to the first primer pair is shown in SEQ ID NO.4, and the nucleotide sequence of the crRNA corresponding to the second primer pair is shown in SEQ ID NO.8.

[0016] A kit for sex identification of poplar trees, the kit being suitable for sex identification of American black poplar and / or European and American poplar, and comprising any of the aforementioned detection systems.

[0017] A poplar sex visualization detection method using any of the aforementioned detection systems, the method being used for sex identification of American black poplar and / or European and American poplar, comprising the following steps:

[0018] (1) Extract genomic DNA from the poplar trees to be tested;

[0019] (2) Using the DNA from step (1) as a template, perform RPA amplification using the primer pair and probe;

[0020] (3) Dilute the amplification product and add it dropwise to the side-flow chromatography test strip. Observe after the reaction is complete. If both the control line (C line) and the detection line (T line) are colored, it is determined to be male; if only the control line is colored, it is determined to be female.

[0021] In some embodiments, the RPA amplification in step (2) is performed at 37-42°C.

[0022] A method for visually detecting the sex of poplar trees based on the aforementioned detection system, the method being used for sex identification of American black poplar and / or European and American poplar, comprising the following steps:

[0023] (1) Extract genomic DNA from the poplar trees to be tested;

[0024] (2) Using the DNA from step (1) as a template, perform RPA amplification using the primer pair;

[0025] (3) Mix the amplification product with a system containing Cas 12a protein, crRNA corresponding to the primer pair used, and a double-labeled reporter molecule;

[0026] (4) Add the reaction product to the side-flow chromatography test strip and observe after the reaction is complete; if both the control line and the detection line show color, it is determined to be male; if only the control line shows color, it is determined to be female.

[0027] The application of any of the detection systems or kits described herein, or any of the methods described herein, in the early screening of non-fluffy male poplars of American black poplar and / or European and American poplars, or in the sex identification of breeding materials.

[0028] Compared with the prior art, this application has the following beneficial effects:

[0029] 1) This invention develops a poplar sex visualization detection primer, probe, and crRNA based on RPA-LFD and RPA-CRISPR / Cas 12a, which can achieve sex identification in the seedling stage before flowering. Detection is performed using test strips, providing intuitive and reliable results without relying on plant morphology or sophisticated instruments. This significantly reduces the time required for sex identification, avoids the problem of later pollen contamination caused by the mixing of female plants in breeding or afforestation, and significantly improves poplar breeding efficiency and afforestation quality.

[0030] 2) The primers of this invention are designed based on the MSL sex-determining gene sequence of poplar. The further screened primer pairs have high amplification efficiency and strong specificity, and can be stably applied in poplar. The detection process only requires a constant temperature device, is simple to operate, and has a short time, making it suitable for on-site detection and easy to promote and apply in breeding bases and forestry production units.

[0031] 3) This invention constructs two visualization detection systems based on RPA-LFD and RPA-CRISPR / Cas 12a, both of which can quickly and intuitively identify the sex of poplar trees, providing a more convenient detection method for nurseries and forest tree breeding.

[0032] 4) This invention enables early, rapid, and visual screening of male poplar trees, reducing the probability of female trees being mixed into afforestation or breeding populations from the source. This effectively avoids pollutant pollution from mature female trees and reduces the impact of pollen on the urban environment, green landscapes, and municipal facilities (such as drainage systems). Simultaneously, it accelerates the breeding process of pollen-free poplars, promoting the development of forestry production towards an eco-friendly direction, and possessing significant ecological and economic benefits. Attached Figure Description

[0033] Figure 1 Agarose gel electrophoresis image of the RPA amplification products of the 5 primer pairs designed in a known male, M is the marker;

[0034] Figure 2 The figures show the results of poplar sex visualization based on RPA-LFD. A shows the test strip results for sex identification of 10 hybrid progeny materials of American black poplar and European poplar amplified using primer pair RPA-MSL-5F / 5R; B shows the test strip results for sex identification of 10 natural population materials of American black poplar amplified using primer pair RPA-MSL-6F / 6R2. In the figures, "PC" and "NC" represent positive control and negative control, respectively (control samples were taken from poplar plants with known sex on campus).

[0035] Figure 3 The image shows the results of the sex visualization detection of poplar trees based on RPA-CRISPR / Cas 12a. A shows the test strip detection results of sex identification of 10 hybrid progeny materials of American black poplar and European poplar amplified by primer pair RPA-MSL-5F / 5R. B shows the test strip detection results of 10 natural population materials of American black poplar amplified by primer pair RPA-MSL-6F / 6R2. In the figure, "PC" and "NC" represent positive control and negative control, respectively (control samples were taken from poplar plants with known sex on campus). Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional molecular biology methods; the materials and reagents used are commercially available unless otherwise specified.

[0038] The hybrid populations and natural populations of American black poplar used in Examples 2 and 3 below were all taken from germplasm resources preserved by our research group. All plant materials used were young leaves collected and stored in a -80℃ freezer.

[0039] Information on the poplar trees used in the following examples is shown in Table 1.

[0040] Table 1. All poplar population samples tested.

[0041]

[0042] Detailed information on the primers, probes, and crRNA sequences used in the RPA-LFD and RPA-CRISPR / Cas 12a reactions is shown in Table 2.

[0043] Table 2 Primer, probe, and crRNA sequences designed for visual detection of poplar sex based on RPA-LFD and RPA-CRISPR / Cas 12a.

[0044]

[0045] Example 1: Establishment of a Visualized Poplar Sex Detection Method Based on RPA-LFD and RPA-CRISPR / Cas 12a

[0046] 1.1 RPA reaction primer screening

[0047] Based on the reported poplar MSL sex-determining gene sequence and considering the technical requirements for primer design in RPA isothermal amplification reactions, five pairs of candidate RPA primers were designed. The primers were 28–35 nucleotides in length, with a GC content controlled between 30% and 70%, and avoided palindromic structures, continuous single-base repeats, and stable internal secondary structures. The specificity, reproducibility, and specificity of each candidate primer were evaluated through RPA amplification screening experiments. The RPA primer combinations that effectively amplified only male poplar DNA, produced clear, single-target bands, and exhibited stable and consistent experimental results, with reliable identification ability, were selected for use in the construction of subsequent detection systems. The RPA reaction system consisted of: one lyophilized RT-RPA / RAA 4.0 (basic type) nucleic acid amplification reagent (purchased from Suzhou Keer Life Science Technology Co., Ltd.), 2 μL of positive DNA template, 1 μL each of forward and reverse primers (10 μM), 1.25 μL of magnesium acetate (280 mM), and the remainder was brought to a total of 25 μL with ddH2O. The reaction was carried out at the recommended temperature of 42℃ in a metal bath according to the kit instructions. After the reaction, the product was extracted with a chloroform-isoamyl alcohol mixture (24:1), and 7 μL of the extract was analyzed by 1.5% agarose gel electrophoresis. The electrophoresis results were observed. Figure 1 As shown, all five primer pairs amplified positive electrophoretic bands. Primers 3 and 5 showed the best amplification results, and these two pairs were selected for subsequent experiments. Primers 3 were named RPA-MSL-5F and RPA-MSL-5R, with nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. Primers 5 were named RPA-MSL-6F and RPA-MSL-6R2, with nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0048] 1.2 Establishment of a Visualized Poplar Sex Detection Method Based on RPA-LFD

[0049] According to the primer and probe design requirements of the RPA-LFD detection method, based on the two primer pairs screened in 1.1 above, a modified gene marker, biotin, was added to the 5' end of the upstream primer. The 5' end of the probe was labeled with an antigen marker, FITC; the probe contained a base nucleotide analog replacing the nucleotide, tetrahydrofuran residue (THF), to improve hybridization stability; and the 3' end of the probe contained a polymerase extension blocking group, C3-spacer, to prevent non-specific extension. The probe sequences designed for the two primer pairs are shown in SEQ ID NO.3 and SEQ ID NO.7.

[0050] Using the modified primers and probes described above, as well as RT-RPA / RAA4.0 (fluorescent) nucleic acid amplification reagent, poplar-specific genomic fragments were amplified.

[0051] The RPA reaction system consisted of: one fluorescent lyophilized pellet, 2 μL of DNA template, 1 μL each of upstream and downstream primers RPA-MSL-F / R (10 μM), 0.25 μL of NFO-Probe (10 μM), 1.25 μL of magnesium acetate (280 mM), and water to a final volume of 25 μL. The amplification reaction conditions were: 42℃ for 20 min.

[0052] After the RPA reaction is complete, the amplification product is thoroughly mixed with 1 mL of sterile water to dilute it, and then tested using an LFD test strip. Visual observation is performed after 3-5 minutes. If both the control line and the test line are visible on the test strip, the sample is male; if only the control line is visible and no test line is visible, the sample is female.

[0053] 1.3 Establishment of a Visualized Poplar Sex Detection Method Based on RPA-CRISPR / Cas 12a

[0054] Based on the locations of the two selected primer pairs, a crRNA specifically targeting the PAM site of the Cas 12a endonuclease was designed. The single-stranded DNA reporter probe was FB Reporter, with the sequence 6-FAM-TCCCCTCCCCTCCCC-biotin.

[0055] Poplar-specific genomic fragments were amplified using the primer pairs selected in 1.1 above and RT-RPA / RAA 4.0 (basic) nucleic acid amplification reagent.

[0056] The RPA reaction system consisted of: one basic lyophilized pellet, 2 μL of DNA template, 1 μL each of upstream and downstream primers RPA-MSL-F / R (10 μM), 1.5 μL of magnesium acetate (280 mM), and water to a final volume of 25 μL. The amplification conditions were: 42℃ for 15 min, followed by inactivation at 65℃ for 5 min.

[0057] Add 2 μL of the RPA reaction product diluted 10-fold to a CRISPR / Cas 12a detection system (total reaction volume 20 μL, including 0.25 μL Cas 12a (2500 ng / μL), 2 μL 10×RNase-free NEB2.1 Buffer, 0.5 μL crRNA (200 nM), 0.1 μL reporter probe FB (50 nM), and 15.15 μL DEPC-H2O). Incubate at 37℃ for 20 min, followed by inactivation at 70℃ for 5 min. After the reaction, thoroughly mix the reaction product with 80 μL of sterile water to dilute the product and use a test strip for detection. Visually observe after 3-5 min. If the test strip shows both the control line and the test line, the sample is male; if the test strip only shows the control line and not the test line, the sample is female.

[0058] Example 2: Application of a Visualized Poplar Sex Detection Method Based on RPA-LFD

[0059] Genomic DNA from 10 individuals from hybrid families of American black poplar and European poplar, and 10 individuals from a natural population of American black poplar, were selected for detection (see Table 1). Materials from the hybrid families of American black poplar and European poplar were amplified using primer pair RPA-MSL-5F / 5R; materials from the natural population of American black poplar were amplified using primer pair RPA-MSL-6F / 6R. Control samples were all obtained from sex-known poplar plants in the laboratory. All tested poplar DNA was diluted to a suitable detection range before RPA reaction, and its specificity was detected using LFD test strips. All samples were detected according to the RPA-LFD-based poplar sex visualization detection method and operating procedures described in Example 1.2.

[0060] The RPA-LFD system results showed that the RPA amplification reaction of male poplar genomic DNA showed double bands (detection line and control line), while female poplar only showed a control line band with no detection line (see [link]). Figure 2 ).

[0061] Example 3: Application of a Visualized Poplar Sex Detection Method Based on RPA-CRISPR / Cas 12a

[0062] Materials from the same poplar species as in Example 2 were selected for testing (see Table 1). Genomic DNA from 10 individuals in the hybrid offspring families of American black poplar and European poplar was amplified using the RPA-MSL-5F / 5R primer pair; genomic DNA from 10 individuals in the natural population of American black poplar was amplified using the RPA-MSL-6F / 6R primer pair. Control samples were all obtained from sex-known poplar plants in the laboratory. All tested poplar materials were diluted to a suitable detection range before RPA amplification, following the RPA reaction procedure described in Example 1.3. The amplified products were added to the Cas12a detection system as described in Example 1.3 and interpreted using lateral flow chromatography strips.

[0063] The results of RPA-CRISPR / Cas 12a system analysis showed that the RPA amplification reaction of genomic DNA from male poplar populations showed double bands (detection line and control line), while female poplars only showed a control line band with no detection line (see [link]). Figure 3 ).

[0064] In summary, this invention provides a visual detection primer, probe, and crRNA for poplar sex based on RPA-LFD and RPA-CRISPR / Cas 12a. It can be used for sex identification in F1 generation and can also be applied to the early identification of sex in European and American black poplar. It has important applications in sex detection of European and American black poplar seedlings, selection breeding of non-fluffy poplars, and management of poplar fluff.

[0065] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A recombinase polymerase amplification detection system for gender identification of poplar, characterized in that, The system is suitable for gender identification of Populus deltoides and / or Populus deltoides x Populus nigra, and comprises: (a) a first primer pair consisting of a nucleotide sequence as shown in SEQ ID NO. 1 and a nucleotide sequence as shown in SEQ ID NO. 2; and / or a second primer pair consisting of a nucleotide sequence as shown in SEQ ID NO. 5 and a nucleotide sequence as shown in SEQ ID NO. 6; (b) a detection module used in cooperation with the primer pair in (a), the detection module being selected from any one of the following: (i) a lateral flow chromatographic test strip detection module; or (ii) a CRISPR / Cas 12a signal output detection module.

2. The detection system of claim 1, wherein, When the detection module is a lateral flow chromatographic test strip detection module, the 5' end of the nucleotide sequence as shown in SEQ ID NO. 1 and / or SEQ ID NO. 5 is covalently linked to a first reporter molecule, and the system further comprises a probe corresponding to the primer pair, the 5' end of the probe being covalently linked to a second reporter molecule different from the first reporter molecule.

3. The detection system of claim 2, wherein, The first reporter molecule is biotin, and the second reporter molecule is fluorescein; and the detection line of the lateral flow chromatographic test strip is coated with an antibody against the second reporter molecule.

4. The detection system of claim 3, wherein, The nucleotide sequence of the probe is as shown in SEQ ID NO. 3 or SEQ ID NO. 7, and it internally comprises a tetrahydrofuran residue and a 3' end extension blocking group.

5. The detection system of claim 1, wherein, When the detection module is a CRISPR / Cas 12a signal output detection module, the system further comprises a crRNA corresponding to the primer pair, wherein the nucleotide sequence of the crRNA corresponding to the first primer pair is as shown in SEQ ID NO. 4, and the nucleotide sequence of the crRNA corresponding to the second primer pair is as shown in SEQ ID NO.

8.

6. A kit for gender identification of Populus, characterized by, The kit is suitable for gender identification of Populus deltoides and / or Populus deltoides x Populus nigra, and comprises the detection system of any one of claims 1-5.

7. A visual detection method of gender of poplar based on the detection system according to any one of claims 2 to 4, characterized in that, The method is used for gender identification of Populus deltoides and / or Populus deltoides x Populus nigra, comprising the following steps: (1) extracting genomic DNA of the tested poplar; (2) using the primer pair and the probe to perform RPA amplification with the DNA of step (1) as a template; (3) adding the amplification product to a lateral flow chromatographic test strip, and observing after the reaction is completed; if both the quality control line and the detection line develop color, it is determined to be male; if only the quality control line develops color, it is determined to be female.

8. The method of claim 7, wherein, The RPA amplification of step (2) is performed at 37-42°C.

9. A method for visualizing the gender of a poplar tree based on the detection system of claim 5, characterized in that, The method is used for gender identification of Populus deltoides and / or Populus deltoides x Populus nigra, comprising the following steps: (1) extracting genomic DNA of the tested poplar; (2) using the primer pair to perform RPA amplification with the DNA of step (1) as a template; (3) mixing the amplification product with a system comprising Cas 12a protein, crRNA corresponding to the primer pair used, and double-labeled reporter molecules for reaction; (4) adding the reaction product to a lateral flow chromatographic test strip, and observing after the reaction is completed; if both the quality control line and the detection line develop color, it is determined to be male; if only the quality control line develops color, it is determined to be female.

10. Use of the detection system or kit of any one of claims 1 to 6, or the method of any one of claims 7 to 9, in early screening or sex identification of breeding material of Populus deltoids and / or Populus deltoids x Populus nigra male sterile clones.

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