A two-way screening method for pepper haploid identification, a screening system and application thereof

Abstract

The present application relates to a kind of two-way screening method for pepper haploid identification, screening system and its application, belong to plant molecular breeding and biotechnology field.The screening system includes root-specific expression visual color module and constitutive promoter driven expression codA negative screening module.The system is constructed in pepper haploid induction line, and the offspring is obtained by hybridization of the induction line and target material, and candidate haploid can be obtained by seedling root color observation combined with 5-FC treatment, which is survival and root colorless seedling.The method is suitable for pepper small grain, seed coat lignification, and it is difficult to identify haploid using traditional phenotype method, with the advantages of high screening accuracy, high flux, low cost and easy to standardize promotion.Experiments show that in about 4000 hybrid progeny seeds, 20 pepper plants confirmed as haploid by molecular detection and flow cytometry.

Description

Technical Field

[0001] This invention belongs to the field of plant haploid identification technology, specifically relating to a two-way screening method, screening system and its application for haploid identification of pepper. Background Technology

[0002] Haploid breeding is a highly efficient modern biological breeding strategy. By inducing hybridization into haploids through haploid inducing lines, and then obtaining homozygous double haploid materials through natural or artificial doubling, the breeding cycle can be significantly shortened and breeding efficiency improved.

[0003] Currently, the overall haploid induction rate is low. The haploid induction rate in maize is typically 5–10%, while in other species it is usually only 0.5–2%, for example, the induction rate in pepper is about 0.6%. At this level of induction rate, without efficient screening methods, it is necessary to identify each plant individually among a large number of hybrid offspring, which is both labor-intensive and costly.

[0004] Currently, conventional methods for haploid identification mainly include phenotypic observation, molecular marker detection, chromosome counting, and flow cytometry. However, these methods have the following shortcomings in chili peppers: 1. Chili seeds are small, making it difficult to visually select them based on differences in appearance at the seed stage; 2. The seed coat of chili peppers is significantly lignified, which is not conducive to the observation of traditional seed markers; 3. Individual PCR testing or flow cytometry identification is costly and has low throughput, making it difficult to meet the needs of large-scale breeding applications; 4. Single phenotypic markers often carry the risk of false negatives or missed screenings, limiting their accuracy.

[0005] Therefore, developing a high-throughput, low-cost, and high-accuracy screening technology suitable for haploid-induced progeny in peppers is a pressing technical problem to be solved in this field. This invention addresses the aforementioned problems in existing pepper haploid screening methods by providing a high-throughput, high-accuracy, and easy-to-operate two-way screening method. This method effectively solves the shortcomings of traditional screening methods, such as complex operation, high cost, and low accuracy, and is suitable for large-scale application in pepper haploid breeding. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a two-way screening method for identifying haploid peppers, which can effectively overcome the defects of traditional screening methods such as complex operation, high cost, and low accuracy, and is suitable for large-scale application in haploid pepper breeding.

[0007] The technical solution of this invention mainly includes the following: This invention first provides a two-way screening system for screening haploid peppers, the two-way screening system comprising: (1) First screening module: a root-specific expression visualization colorimetric module, used to generate a recognizable color signal in the roots of pepper seedlings carrying the module; preferably, the visualization colorimetric module is a RUBY colorimetric module, which can produce betalains in the root tissue, making the roots appear red or reddish-purple.

[0008] (2) Second screening module: negative screening module, including a codA gene expressed by a constitutive promoter; the cytosine deaminase encoded by codA catalyzes the conversion of 5-FC into cytotoxic 5-FU in the presence of 5-fluorocytosine (5-FC), thereby inhibiting the germination and growth of individuals carrying this module.

[0009] The bidirectional screening system is configured in the chili haploid induction line material, so that in the offspring obtained after the induction line is hybridized with the target chili material, non-haploid individuals show root coloration and / or cannot germinate or grow normally under 5-FC treatment, while haploid individuals show no root coloration and can survive normally under 5-FC treatment.

[0010] Preferably, the first screening module is driven by a root-specific promoter, and the expression box sequence of the visualization and colorimetric module is shown in SEQ ID NO.1. The expression box sequence of the negative screening module is shown in SEQ ID NO.2.

[0011] Furthermore, the first and second screening modules of the bidirectional screening system are integrated into the same plant expression vector, or integrated into different vectors and then genetically aggregated into the same pepper haploid inducible line. Preferably, the first and second screening modules are integrated into the same vector to ensure the consistency rate of the two screenings and the true haploid confirmation rate.

[0012] The relative position, arrangement direction, whether the root chromogenic module and the codA negative screening module are located within the same T-DNA region, and whether they are configured with independent transcriptional regulatory units, all of these factors can affect the expression stability and screening accuracy of the screening system of this invention. This invention integrates the RUBY chromogenic module and the codA negative screening module into the same T-DNA region, and their expression is driven by independent promoters. In one specific embodiment, they are arranged in a relatively reversed manner, and each has its own termination sequence, thereby reducing the risk of transcriptional cross-reading and mutual interference.

[0013] Preferably, this invention uses a single T-DNA integration vector to construct a bidirectional screening system. The vector includes a root-specific promoter-driven RUBY chromogenic module, a constitutive promoter-driven codA negative screening module, a plant selection marker module, and left and right boundaries of the T-DNA. The vector structure is: LB–selection marker–Proot-RUBY-Tnos–P35S-codA-T35S–RB. The vector can be constructed using homologous recombination, Golden Gate, or other methods, verified by sequencing, and then transformed into Agrobacterium and further transformed into background material of a pepper haploid inducible line.

[0014] This invention also provides a method for screening haploids in chili peppers, comprising the following steps: S1. Provide a chili haploid induction line material carrying the bidirectional screening system, and use it as the female parent and the target chili material as the male parent to crossbreed to obtain offspring seeds; S2. Germinate the offspring seeds and observe the root coloration after the seedling roots reach the preset length. S3. Place the germinating seedlings under a treatment containing 5-FC for negative screening; S4. Select seedlings that survived normally after 5-FC treatment and whose roots did not show coloration as candidate haploids; S5. Perform molecular detection and / or ploidy identification on the candidate haploids to obtain confirmed pepper haploids.

[0015] Preferably, the seedling root length in step S2 is 3-8 cm, more preferably about 5 cm.

[0016] Preferably, the concentration of 5-FC in step S3 is not less than 0.5 g / L, more preferably 0.5 to 1.5 g / L, more preferably 0.4 to 0.8 g / L, and most preferably 0.5 g / L.

[0017] Preferably, the processing time in step S3 is 3 to 7 days, more preferably 5 days.

[0018] Preferably, the germination culture adopts a filter paper germination system, a hydroponic system, an agar culture system, a sponge seedling system, or a combination thereof.

[0019] Preferably, the molecular detection in step S5 is PCR detection targeting the RUBY chromogenic module and / or the codA gene, and the ploidy identification is flow cytometry detection, chromosome detection, or a combination thereof.

[0020] The beneficial effects of this invention are: (1) The root-specific colorimetric screening is combined with the codA / 5-FC negative screening to form a dual judgment mechanism and improve the accuracy of pepper haploid screening. (2) Make full use of the natural colorless characteristics of pepper seedling roots and set the observation window in the root tissue, which is suitable for the characteristics of pepper seeds being small, with lignified seed coats, and not easily identifiable through the seed stage; (3) Through front-end dual screening, the number of samples for subsequent molecular detection and flow cytometry detection can be significantly reduced, thereby reducing screening costs; (4) This method can process a large number of hybrid offspring seeds in batches, and is suitable for large-scale breeding applications; (5) Examples demonstrate that the present invention can rapidly and accurately obtain 20 haploid plants confirmed by molecular detection and flow cytometry from approximately 4,000 hybrid offspring seeds, and has good practical value. Attached Figure Description

[0021] Figure 1 Root colorimetric comparison chart.

[0022] Figure 2 : Schematic diagram of the carrier structure. Detailed Implementation

[0023] To facilitate a clearer understanding of the technical content of this invention by those skilled in the art, the invention will be further described below in conjunction with specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The pepper haploid induction line used in this invention is a gene-edited material obtained by knocking out the pepper haploid induction-related gene (DMP homolog) using CRISPR / Cas technology. A bidirectional screening system is then introduced into this CRISPR-edited background material to form a pepper induction line possessing both haploid induction and screening capabilities.

[0026] Example 1: Construction of bidirectional screening induction line materials 1. Experimental Objective Construct a haploid induction line for pepper that simultaneously carries a root-specific chromogenic module and a codA negative sieve module.

[0027] 2. Carrier Design Construct a plant expression vector, wherein the vector contains at least the following two expression modules: (1) Root chromogenic module: The root-specific promoter is used to drive the expression of the RUBY chromogenic module, so that betalains accumulate in the root tissue of pepper seedlings and appear red or reddish-purple; the sequence of the root-specific RUBY expression box is shown in SEQ ID NO.1.

[0028] (2) Negative screening module: The codA gene expression is driven by a constitutive promoter, the expression cassette sequence of which is shown in SEQ ID NO.2, and the coda protein cds sequence is shown in SEQ ID NO.3. The cytosine deaminase encoded by codA does not significantly affect the normal growth and development of plants in the absence of 5-FC. In the presence of 5-FC, it converts 5-FC into a cytotoxic product, thereby inhibiting the cell growth of individuals carrying this gene.

[0029] In this embodiment, the two modules described above are constructed in the same vector. Those skilled in the art can also construct the two modules in different vectors and then introduce them into the same material via genetic polymerization.

[0030] 3. Genetic transformation The aforementioned expression vector was introduced into the background material of the pepper haploid inducible line using Agrobacterium-mediated transformation.

[0031] 4. Screening and identification of positive materials PCR identification was performed on the regenerated plants after genetic transformation to detect whether the RUBY module and codA gene were integrated; the transgene copy number was further analyzed by conventional methods such as qPCR and Southern blot to screen for single-copy materials; the obtained materials were continuously self-crossed to screen for single-copy homozygous transgene lines; the chromogenic phenotype of the seedling roots was observed and the negative screening response under 5-FC treatment was detected.

[0032] 5. Results A single-copy homozygous pepper haploid induction line material carrying both a root-specific RUBY colorimetric module and a constitutive codA negative screening module was obtained. Its root coloration is stable and sensitive to 5-FC treatment, and it can be used for subsequent pepper haploid screening.

[0033] Example 2: Concentration Optimization of 5-FC Negative Screen 1. Experimental Objective Optimize the concentration of 5-FC treatment suitable for the germination and seedling stage of peppers.

[0034] 2. Experimental Materials Wild-type pepper seeds, transgenic pepper seeds carrying a constitutive codA module, 5-FC reagent, filter paper, petri dishes, and sterile water.

[0035] 3. Experimental Methods Select uniformly sized, plump chili seeds for germination. Seeds can be surface-sterilized first: treat with 75% (w / v) ethanol for 30 seconds, then with 1% (w / v) sodium hypochlorite for 5–10 minutes, and finally rinse with sterile water 3–5 times.

[0036] Spread the seeds evenly in a petri dish or germination box lined with filter paper, add an appropriate amount of sterile water to keep them moist, and place them at 25±2℃ for germination culture. After the seeds germinate uniformly, replace the solution with different concentrations of 5-FC and treat continuously for 5 days, then record the survival rate and growth status.

[0037] 4. Handling Design and Results (1) Wild-type material: The concentrations of 5-FC were set at 0.25 g / L, 0.5 g / L, 1.0 g / L and 5.0 g / L, respectively. After 5 days of treatment, the survival rate of wild-type pepper seedlings was 98%.

[0038] (2) Transgenic materials: The concentrations of 5-FC were set at 0.25 g / L, 0.5 g / L, 1.0 g / L and 1.5 g / L, respectively. After 5 days of treatment, the survival rates of transgenic seedlings were 90%, 5%, 7% and 7%, respectively.

[0039] 5. Results Analysis The results showed that 5-FC had extremely low toxicity to wild-type pepper seedlings, but significantly inhibited the growth of transgenic materials carrying the codA module. 0.5 g / L significantly reduced the survival rate of transgenic materials while having minimal impact on wild-type materials; therefore, 0.5 g / L was determined to be the optimal negative sieve concentration.

[0040] Example 3: Dual screening of haploid-induced progeny of pepper 1. Experimental Objective The bidirectional screening system of this invention is used for high-throughput screening of haploid-induced progeny of peppers.

[0041] 2. Experimental Materials The female parent, a haploid induction line for chili peppers, carries a root-specific RUBY chromogenic module and a constitutive codA negative sieve module; the male parent, a cultivated chili pepper, is a cayenne pepper. 3. Hybridization and Seed Harvesting Artificial hybridization was carried out using a haploid chili induction line carrying a two-way selection system as the female parent and chili pepper (Zanthoxylum bungeanum) as the male parent. After the fruit matured, the seeds were harvested, yielding approximately 4,000 F1 offspring seeds.

[0042] 4. Dual screening process (1) Seed germination: Spread F1 seeds flat on moist filter paper and add water for germination culture. After about 1 week, the seedling roots will be about 5 cm long.

[0043] (2) Root color observation: Observe the color of the seedling roots. Seedlings with red or reddish-purple roots are identified as non-target individuals carrying the induction line color module; seedlings without red roots are retained as candidate materials.

[0044] (3) 5-FC negative sieve treatment: The water in the culture system was replaced with a treatment solution containing 0.5 g / L 5-FC, and the treatment was carried out for 5 consecutive days. Non-haploid individuals carrying the codA module showed growth arrest, root browning and necrosis, and hypocotyl shrinkage or death; candidate haploids without the module were able to survive normally.

[0045] (4) Obtaining candidate haploids: 130 surviving seedlings were obtained after 5-FC treatment; among them, 20 pepper seedlings without red roots were further screened.

[0046] 5. Candidate haplotype verification (1) Molecular detection: Genomic DNA was extracted from 20 candidate seedlings and PCR was performed using specific primers for the RUBY module and codA module. The results showed that no corresponding transgenic bands were detected.

[0047] (2) Flow cytometry detection: Fresh tissue samples from 20 candidate materials were used to prepare cell nuclear suspensions, which were then stained with DNA dyes and analyzed by flow cytometry. The results showed that all 20 materials exhibited haploid peaks.

[0048] 6. Results All 20 seedlings were confirmed as haploid pepper materials. This demonstrates that the two-way screening system established in this invention can accurately and efficiently screen haploid individuals from a large number of offspring.

[0049] Example 4: Establishing a standardized screening process 1. Seed pretreatment Take mature, plump, and undamaged seeds obtained from hybridization in Example 3, disinfect their surfaces with 75% ethanol and sodium hypochlorite, and then rinse them with sterile water for later use.

[0050] 2. Germination Culture Line a petri dish or germination box with filter paper, moisten it with sterile water, and evenly spread the seeds on the filter paper. Incubate at 25±2℃ under 16 h light / 8 h dark conditions. Observe and replenish water daily.

[0051] 3. Observation of root color development When the seedling roots reach a length of 3–8 cm, observe the root color. Record the color intensity as “+++”, “++”, “+”, and “-”. Individuals marked with “-” will proceed to the subsequent negative screening step.

[0052] 4. 5-FC processing Add 0.5 g / L 5-FC working solution to the germination system and treat continuously for 5 days, replenishing the working solution every 1-2 days to maintain a stable concentration. After treatment, count the number of normally surviving, severely inhibited, and dead individuals.

[0053] 5. Candidate haplotype verification DNA was extracted from surviving seedlings without red roots for PCR detection, and ploidy analysis was performed using flow cytometry to confirm haploid materials.

[0054] Example 5: The Influence of Different Vector Construction Methods on Screening Results This invention (Example 1): Same vector, same T-DNA, root-specific promoter -RUBY+ constitutive-codA Comparative Example 1: Same carrier, but RUBY is driven by a constitutive promoter 35S. Comparative Example 2: Two vectors were constructed separately and then hybridized into the same material. Each of the above schemes was used as the female parent and crossed with the same male parent of chili pepper, yielding approximately 1000 offspring seeds for each scheme.

[0055] Screening process: Observe root color development on day 7; treat with 0.5 g / L 5-FC for 5 days; count survival, color development, and candidate numbers.

[0056] The results showed that in Example 1, the root coloration clarity rate reached 94.6%, the background interference rate was only 6.8%, the negative screening mortality rate of non-haploids under 5-FC treatment was 95.2%, and the final true haploid confirmation rate among candidate seedlings was 88.9%. In contrast, in Comparative Example 1, due to 35S-driven RUBY causing background coloration in the hypocotyl and cotyledons, the background interference rate increased to 31.5%, and the final true haploid confirmation rate decreased to 42.4%. Although the root coloration in Comparative Example 2 was relatively clear, the coexistence stability of the two modules in the offspring decreased because the RUBY module and the codA module belong to different genetic units, the double screening consistency rate dropped to 76.2%, and the final true haploid confirmation rate was only 31.7%.

[0057] Table 1

[0058] Root color clarity definition: The proportion of seedlings whose roots can be clearly identified as red or reddish-purple at a set observation time point (e.g., 7 days after germination, when the root length is about 5 cm).

[0059] Background interference rate is defined as the proportion of seedlings in which tissues other than the roots (such as hypocotyls, cotyledons, and seed coat residues) exhibit obvious color signals that affect the judgment.

[0060] 5-FC negative screening mortality rate definition: the proportion of individuals carrying the negative screening module who die or show obvious growth arrest after being treated with 0.5 g / L 5-FC for 5 days.

[0061] Double-screen consistency rate definition: The proportion of colorimetric judgment results that are consistent with the 5-FC negative screening results.

[0062] Number of candidate seedlings: The number of candidate haploid seedlings after double screening.

[0063] Final haploid confirmation rate is defined as the proportion of true haploids among candidate seedlings after PCR confirmation.

[0064] The reason why this invention is superior to Comparative Example 1 is: Root-specific expression concentrates the red color in the root, making it easy to identify; constitutive promoter-driven 35S-RUBY also shows color in the hypocotyl, cotyledons and other parts, interfering with the judgment; high background interference leads to an increase in false positives and an inflated number of candidates, but the true haploid confirmation rate decreases.

[0065] The reason why this invention is superior to Comparative Example 2 is: The same T-DNA can ensure that both modules are delivered together; after the separate construction, polymerization is prone to separation, and the progeny may only carry RUBY or only carry codA; causing inconsistency in double screening, affecting the enrichment effect and accuracy.

[0066] Example 6 This invention (Example 1): Same vector, same T-DNA, root-specific promoter -RUBY+ constitutive-codA Comparative Example 3: Root-specific promoter -GFP + 35S-codA Comparative Example 4: Rootspecific promoter -GUS + 35S-codA The same haploid induction line for peppers was used as the background; the same male parent was used; each group had approximately 800-1000 offspring seeds; the same observation time point and the same 5-FC negative screening conditions were used.

[0067] The results showed that when using RUBY as the visualization and colorimetric module, a clear and stable red signal could be observed in the seedling roots under natural light, with a root signal clarity rate of 93.8%. No additional substrate or specialized equipment was required, making it suitable for rapid screening in large-scale populations. The final haploid confirmation rate among candidate materials reached 90.0%. In contrast, when using GFP as the colorimetric marker, although the signal could be observed under fluorescence equipment, specialized instruments were required, and some samples experienced background fluorescence interference, resulting in a final haploid confirmation rate of 57.1%. When using GUS as the colorimetric marker, substrate staining was necessary, and the staining process was destructive to live seedlings, which was not conducive to subsequent seedling preservation and continuous screening, resulting in a final haploid confirmation rate of 62.5%.

[0068] Table 2

[0069] The above description is only a partial embodiment of the technical solution of the present invention. These embodiments are merely illustrative examples intended to help those skilled in the art understand the core concept of the present invention, and are not exhaustive enumerations of all embodiments of the present invention. The scope of protection of the present invention should be determined by the claims. Any adaptive modifications, equivalent substitutions of technical features, further optimizations and improvements made based on the basic principles and concepts disclosed in the present invention are all within the scope of protection of the present invention.

Claims

1. A two-way screening system for screening haploid peppers, characterized in that, The two-way screening system includes a first screening module and a second screening module; The first screening module is a root-specific expression visualization colorimetric module, which is used to generate a recognizable color signal in the roots of pepper seedlings carrying the module; the visualization colorimetric module is a RUBY colorimetric module; the first screening module is expressed by a root-specific promoter, and the expression box sequence of the visualization colorimetric module is shown in SEQ ID NO.1; The second screening module is a negative screening module, which includes the codA gene encoding cytosine deaminase expressed by a constitutive promoter; the expression cassette sequence of the negative screening module is shown in SEQ ID NO.2; The first and second screening modules in the bidirectional screening system are integrated into the same plant expression vector; The bidirectional screening system is configured in the chili haploid induction line material and is used to screen haploids in the offspring obtained by hybridization of the chili haploid induction line and the target chili material. In the offspring, non-haploid individuals show root coloration and cannot grow normally under 5-fluorocytosine treatment, while haploid individuals show no root coloration and can grow normally under 5-fluorocytosine treatment.

2. The bidirectional screening system according to claim 1, characterized in that, The chili pepper haploid inducible line material is a single-copy homozygous transgenic material.

3. A method for screening haploid peppers, characterized in that, Includes the following steps: S1. Using the chili haploid induction line material of the bidirectional screening system described in any one of claims 1 to 2 as the female parent and the target chili material as the male parent, hybridize to obtain offspring seeds; S2. Germinate the offspring seeds and observe whether the seedling roots show color after the seedling roots reach the preset length. S3. After germination, the seedlings were subjected to negative screening under treatment conditions containing 5-fluorocytosine. S4. Seedlings that survived treatment with 5-fluorocytosine and whose roots did not show color were selected as candidate haploids. S5. Perform ploidy identification and / or molecular detection on the candidate haploids to obtain pepper haploids.

4. The method for screening haploid peppers according to claim 3, characterized in that, The preset length mentioned in step S2 is 3 to 8 cm.

5. The method for screening haploid peppers according to claim 3, characterized in that, In step S3, the concentration of 5-fluorocytosine is not less than 0.5 g / L.

6. The method for screening haploid peppers according to claim 3, characterized in that, The treatment time for 5-fluorocytosine in step S3 is 5 to 7 days.

7. The application of the bidirectional screening system according to any one of claims 1 to 2 or the pepper haploid screening method according to any one of claims 4 to 6 in the screening of pepper haploid induced progeny.

8. The application according to claim 7, characterized in that, The applications include rapid identification of chili haploids and pre-breeding screening of chili double haploids.

Images