A rapid identification method for brassinosteroid sensitivity of watermelon seedlings

Abstract

The application discloses a kind of watermelon seedling brassinosteroid sensitivity rapid identification method, belong to crop physiological breeding technical field, including S1, seedling treatment: selecting cotyledon not completely unfolded cap period watermelon seedling, 24-epibrassinolide solution is added dropwise at the growth point of seedling;S2, subsection light control culture: under complete darkness condition culture, under weak light condition culture;S3, multi-index comprehensive score: the petiole length of seedling cotyledon, true leaf length and hypocotyl curvature are measured, and comprehensive score S is calculated;S4, sensitivity determination: according to comprehensive score S, the sensitivity of watermelon seedling to brassinosteroid is judged;The method of the application realizes the rapid, accurate, non-destructive identification of watermelon seedling BR sensitivity, and establishes the prediction relationship between seedling BR sensitivity and mature period fruit shape index, provides reliable technical means for early screening of watermelon plant type and fruit shape related traits at seedling stage, and has important application value in watermelon breeding.

Description

Technical Field

[0001] This invention belongs to the field of crop physiological breeding technology and relates to a rapid identification method for the sensitivity of watermelon seedlings to brassinosteroids. Background Technology

[0002] Brassinosteroids (BRs) are important phytosterol hormones that widely participate in regulating key physiological processes in plants, including cell elongation and division, photomorphogenesis, plant architecture shaping, fruit development, and stress response. In crop breeding, BR sensitivity identification is a crucial technique for screening ideal plant architecture materials and conducting targeted improvement. By detecting the degree of plant response to exogenous BRs, germplasm resources with varying BR sensitivity can be effectively distinguished, providing a basis for early seedling selection.

[0003] Currently, research on the BR signaling pathway in model plants such as rice and Arabidopsis thaliana, as well as major crops, is quite in-depth, and related sensitivity detection methods are relatively mature. For example, a BR sensitivity identification method based on measuring the leaf angle after treatment of detached leaf nodes with brassinosteroids has been reported in rice; an indirect identification method based on measuring the hypocotyl length after treatment of seedlings with brassinolide has been reported in Arabidopsis thaliana. However, the above methods are all developed for specific species. As a cucurbitaceous crop, watermelon has significantly different BR signaling pathway components and regulatory mechanisms compared to rice and Arabidopsis thaliana, making it impossible to directly apply the above methods.

[0004] Watermelon is an important economic crop in my country, and its agronomic traits, such as plant architecture (e.g., leaf angle, branching characteristics), fruit shape (fruit shape index), and stress resistance, are all closely related to the BR signaling pathway. However, research on the watermelon BR signaling pathway is relatively lagging, lacking rapid, non-destructive, and high-throughput BR sensitivity detection methods suitable for watermelon seedlings, which severely restricts the development of watermelon BR-related breeding work. On the one hand, breeders cannot efficiently screen BR-sensitive differentially expressed materials during the seedling stage and can only wait for the plants to mature before making indirect judgments through field phenotypes, which is time-consuming and inefficient. On the other hand, the lack of standardized identification methods also hinders the discovery and verification of functional genes in the watermelon BR signaling pathway.

[0005] Therefore, establishing a rapid identification method for BR sensitivity in watermelon is of great significance for watermelon plant type improvement, fruit shape-oriented breeding, and BR signaling pathway research. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid identification method for brassinosteroid (BR) sensitivity in watermelon seedlings. By establishing an identification system using the cotyledon shedding stage as the identification window, micro-drip application at the growing point as the application method, and combined with segmented controlled light cultivation and multi-index weighted scoring, the method achieves rapid, accurate, and non-destructive identification while comprehensively meeting the timeliness, accuracy, and plant survival rate requirements of early seedling screening. This addresses the technical problems of existing BR sensitivity identification methods, such as long identification cycles, single judgment indicators, damage to plants, and ineffective integration of identification and breeding processes. Ultimately, this method improves the efficiency and accuracy of BR sensitivity identification in watermelons, providing a reliable and rapid identification method for early seedling screening of watermelon plant type and fruit shape-related traits.

[0007] The technical solution adopted in this invention is a rapid identification method for the sensitivity of watermelon seedlings to brassinosteroids, the key of which includes the following steps: S1. Seedling treatment: Select watermelon seedlings in the capping stage whose cotyledons have not fully unfolded, and add 5 μL of 0.05 μmol / L to 1 μmol / L 24-epibrassinolide solution to the growing point of the seedling. S2. Segmented controlled light culture: Seedlings treated in step S1 were first cultured in complete darkness for 18-30 hours, and then cultured under low light conditions for 8-16 hours. The light intensity under the above low light conditions was 40 μmol·m⁻¹. -2 ·s -1 ~60 μmol·m -2 ·s -1 ; S3. Multi-index comprehensive score: After the above-mentioned segmented light-controlled culture is completed, the cotyledon petiole length, true leaf length and hypocotyl curvature of the seedlings are measured. The comprehensive score S is calculated based on the measured values ​​or assigned values ​​and the corresponding preset weights. S4. Sensitivity determination: The sensitivity of watermelon seedlings to brassinosteroids is determined based on the comprehensive score S. A value of S higher than the preset threshold is considered highly sensitive, and a value of S not higher than the preset threshold is considered low sensitive.

[0008] The aforementioned "cotyledon-not fully unfolded" cap-removal period refers to the developmental stage after watermelon seed germination, when the cotyledons break through the seed coat but have not yet fully unfolded and flattened. Due to differences in watermelon varieties, seedling temperatures, and seed vigor, the specific timing of the cap-removal period may vary slightly. Those skilled in the art can select an appropriate treatment time within the range of 4 to 8 days after sowing based on the actual emergence situation. As long as the seedlings are in the morphological state of incompletely unfolded cotyledons, the identification effect of this invention can be achieved.

[0009] In step S1, 24-epibrassinolide is used as an example of brassinosteroid active substance. Based on the method principle of this invention, other compounds with brassinosteroid activity, such as brassinolide, 28-homobrassinolide, and 28-epibhomobrassinolide, etc., which are BR activity analogs, can also be expected to achieve similar identification effects under the same concentration range and treatment conditions. Therefore, the 24-epibrassinolide mentioned above in this invention should not be construed as a limitation on the type of active substance. Those skilled in the art can select other sterol compounds with BR activity or their functional equivalents according to actual needs.

[0010] Specifically, the watermelon seedlings in step S1 that have reached the cap removal stage are seedlings obtained by cultivating them in a completely dark environment at 28℃ for 5 days after sowing.

[0011] Specifically, the dropping operation in step S1 is carried out in a low-light environment, where the light intensity is 40 μmol·m⁻¹. -2 ·s -1 ~60 μmol·m -2 ·s -1 In step S1, the 24-epibrassinolide solution may also contain surfactants to reduce the surface tension of the solution and promote its uniform spreading and penetration at the growth point.

[0012] In step S2 of this invention, the segmented light-controlled cultivation adopts a sequential pattern of first darkness and then low light. The core principle is that the darkness stage fully activates the plant's BR signaling pathway, while the low light stage, while maintaining the basic photomorphogenesis of the seedlings and preventing excessive yellowing, amplifies the morphological differences between plants with different BR sensitivities. Based on this principle, while maintaining the core logic of activation in darkness followed by amplification in low light, those skilled in the art can appropriately adjust the specific duration of darkness and low light cultivation according to the tolerance of specific watermelon varieties.

[0013] Preferably, in step S2, the complete darkness incubation time is 24 hours, the low-light incubation time is 12 hours, and the low-light illumination intensity is 50 μmol·m⁻¹. -2 ·s -1 .

[0014] Specifically, the temperature for both the dark culture and the low-light culture was 28°C.

[0015] Furthermore, in step S3, the cotyledon petiole length is the distance from the growing point to the cotyledon leaf surface; the true leaf length is the distance from the growing point to the highest point of the true leaf; the hypocotyl curvature is assigned a value based on the degree of hypocotyl twisting, with a value range of 0 to 1, where 0 is completely upright and 1 is severely twisted.

[0016] Furthermore, the comprehensive score S in step S3 is calculated according to Equation 1: S = 0.4 × a + 0.3 × b + 0.3 × c (Equation 1) In Formula 1, a is the cotyledon petiole length in cm; b is the true leaf length in cm; c is the hypocotyl curvature value, ranging from 0 to 1; 0.4, 0.3, and 0.3 are the preset weights of the corresponding indicators.

[0017] Based on the core idea of ​​this invention, namely, reducing the error of a single indicator through comprehensive evaluation of multiple indicators, those skilled in the art will understand that, under the premise of keeping the above three indicators unchanged, fine-tuning of the weights can still achieve effective differentiation of BR sensitivity, which is a reasonable variation of the comprehensive scoring system of this invention.

[0018] Furthermore, at least 10 watermelon seedlings of known BR-sensitive and BR-weakly sensitive types are taken from the corresponding genetic background, and a comprehensive score S is obtained according to steps S1 to S3. When the S value of all sensitive seedlings is greater than the S value of all weakly sensitive seedlings, the average of the minimum S value among the above sensitive seedlings and the maximum S value among the above weakly sensitive seedlings is used as the preset threshold.

[0019] The above methods are applied in screening watermelon BR signaling pathway mutants and breeding resources with abnormal BR signaling sensitivity.

[0020] Specifically, when the above method is applied to the early screening of watermelon fruit shape, watermelon seedlings with a comprehensive seedling score S lower than the preset threshold under the same genetic background will have rounder fruit shape after maturity, and their fruit shape index will not exceed the fruit shape index threshold. The above-mentioned fruit shape index thresholds are determined in the following way: Take at least 10 watermelon fruits with relatively round and long shapes under the corresponding genetic background, measure the longitudinal and transverse diameters of each fruit, and calculate the fruit shape index SX = longitudinal diameter / transverse diameter. The average of the maximum value of SX for round fruits and the minimum value of SX for long fruits is used as the fruit shape index threshold.

[0021] The rapid identification method for brassinosteroid sensitivity in watermelon seedlings provided by this invention has the following advantages compared with existing BR sensitivity identification techniques: First, this invention significantly shortens the identification cycle. Existing methods require waiting for seedlings to grow to maturity (approximately 50 days) and indirectly inferring sensitivity based on phenotypic observations. This invention can be performed as early as the cotyledon decapitation stage, 5 days after sowing, completing identification within 36 hours, thus greatly shortening the identification cycle. The key to achieving this effect lies in overcoming a long-standing technical bias in the field: the belief that seedlings at the cotyledon decapitation stage are too tender and have poor tolerance to exogenous stimuli, including hormones, making them highly susceptible to damage and deformities during treatment; therefore, operations can only be performed in the late stages of leaf unfolding. This invention successfully overcomes this developmental limitation through the synergistic combination of micro-droplet application at the growth point and segmented controlled light cultivation.

[0022] Secondly, the segmented light-controlled cultivation strategy of this invention effectively solves the technical contradictions in seedling treatment during the decapitation stage. While continuous darkness can fully activate the BR signaling pathway, seedlings will die due to excessive yellowing and etiolation; conversely, premature introduction of light will trigger photomorphogenesis, severely inhibiting BR signal amplification. This invention employs a specific temporal combination of darkness followed by weak light: complete darkness in the early stage fully activates the BR response, while weak light of a specific intensity in the later stage maintains seedling survival while avoiding the inhibition of BR signals by strong light. This maximizes the amplification of phenotypic differences among seedlings with different sensitivities while ensuring complete plant survival. This strategy has not been reported in existing BR sensitivity identification methods.

[0023] Third, this invention significantly improves identification accuracy. Existing methods often use single indicators such as plant height or hypocotyl length as the basis for judgment, resulting in large individual errors and susceptibility to interference from factors such as seed quality and microenvironment. This invention proposes for the first time a weighted comprehensive scoring system based on three indicators: cotyledon petiole length, true leaf length, and hypocotyl curvature. Among them, hypocotyl curvature is an original indicator of this invention. Existing BR sensitivity identification methods mostly focus on the longitudinal elongation of organs (such as hypocotyl length), while this invention discovers for the first time that differences in the strength of BR signal sensitivity induce uneven lateral growth stress in the hypocotyl, manifested as different degrees of hypocotyl torsion. This indicator successfully captures the uneven growth information induced by BR, and the BR response dimension it reflects cannot be provided by a single length measurement. By incorporating curvature into the scoring system, it is possible to effectively distinguish individual plants with similar hypocotyl lengths but different actual BR sensitivities, significantly reducing the rate of missed and false positives. More importantly, the cotyledon petiole, true leaf, and hypocotyl represent the responses of three independent organ systems—cotyledon, newly emerging true leaf, and hypocotyl—to BR, respectively. These three components were not chosen arbitrarily but constitute a three-dimensional assessment system reflecting the continuous developmental process. The weighted synthesis of these three components effectively neutralizes errors caused by the lag in responses of a single organ or individual differences. The accuracy of its representation of BR sensitivity is unattainable by any single or arbitrary combination of indicators.

[0024] Fourth, this invention causes no damage to the plants during implementation, achieving an effective connection between identification and breeding. Existing methods, such as the rice leaf angle method, require cutting detached leaf nodes for soaking, which is destructive sampling; Arabidopsis thaliana BR treatment requires culture on sterile medium, resulting in low transplant survival rates. In contrast, this invention uses a micro-drip method at the growing point, precisely treating the apical meristem with only 5 μL of low-concentration solution. The cotyledons, hypocotyls, and roots are not directly exposed to the agent. After treatment, seedlings can be transplanted normally to the field for continued cultivation and fruiting. This allows for a direct correlation between seedling identification results and mature agronomic traits of the same plant, achieving a seamless connection between the identification and breeding stages.

[0025] Fifth, this invention is the first to discover and verify a strong correlation between the comprehensive BR sensitivity score (S value) of watermelon seedlings and the fruit shape index of mature fruit; that is, plants with an S value below a preset threshold at the seedling stage tend to have rounder fruits. This technical effect was unexpected. In the field of botany, there is no definitive rule for predicting long-term fruit morphology from physiological responses at the seedling stage. Those skilled in the art cannot readily deduce the final expression of this complex quantitative trait, fruit shape, from conventional indicators such as hypocotyl length or leaf angle at the seedling stage. This discovery not only enhances the application value of this invention but also powerfully demonstrates that the identification system established by this invention possesses the depth to reflect the systematic developmental regulation capabilities of plants, which is not possessed by existing single or simple combination identification methods.

[0026] In addition, the present invention is simple to operate, requires only conventional experimental equipment, and its extremely low reagent consumption per plant significantly reduces identification costs, making it particularly suitable for large-scale batch screening in breeding.

[0027] In summary, this invention establishes an innovative identification system that uses the cotyledon capping stage as the identification window, micro-drip application at the growing point as the application method, combined with specific segmented light-controlled culture and comprehensive scoring of three indicators. This system systematically solves the key problems of existing BR sensitivity identification methods, such as long cycle, low accuracy, destructive nature, and high cost. For the first time, it achieves rapid, accurate, and non-destructive identification of watermelon seedling BR sensitivity and has achieved the technical effect of predicting mature fruit shape. It provides a reliable technical means for the early and accurate screening of watermelon plant type and fruit shape related traits. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1This is a schematic diagram illustrating the methods for measuring the length of the cotyledon petiole, the length of the true leaf, and the curvature of the hypocotyl in seedlings. Figure 1 (a) Indicates the location for measuring the length of the cotyledon petiole; Figure 1 (b) Indicates the measurement location for the true leaf length; Figure 1 (c) is a schematic diagram of the standard for assigning curvature values ​​to the hypocotyl.

[0030] Figure 2 This is a comparison of the S values ​​of watermelon seedlings of different genotypes treated with different concentrations of 24-epibrassinolide.

[0031] Figure 3 This is a photograph of the mature fruit from Example 3. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing specific embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Unless otherwise specified in the examples, the standard conditions can be followed; unless the manufacturer of the reagents or instruments used is specified or otherwise not specially described, they are all conventional products that can be purchased commercially.

[0038] It should be noted that the "corresponding genetic background" or "equivalent genetic background" referred to in this invention refers to the specific breeding population targeted for BR sensitivity identification and fruit shape screening. Within this population, the genomic DNA similarity among individuals is high, and the genetic segregation pattern of the target trait is relatively clear. One of the core objectives of breeding work is to aggregate superior traits dispersed in different germplasm resources into the target variety. These germplasm resources may originate from different regions of the world, or even different continents, with vastly different genetic backgrounds. The method provided by this invention does not rely on materials from a specific genotype or region. Instead, it establishes a set of operational standards. For watermelon breeding populations with any genetic background, as long as the S-value threshold and fruit shape index threshold applicable to the population are determined according to steps S1-S3 of this invention and the preset threshold determination method, early seedling screening can be achieved.

[0039] Example 1 This embodiment exemplifies the complete identification process of the method of the present invention, and uses the optimal parameter combination for illustration.

[0040] Experimental preparation: Watermelon seeds were soaked and germinated before being sown in 72-cell trays and cultured in a completely dark environment at 28°C for 5 days.

[0041] S1. Seedling treatment: After the seedlings enter the cotyledon decapitation stage, chemical treatment should be carried out, with a constant concentration of 50 μmol·m⁻¹. -2 ·s -1 Operate in a low-light environment to avoid strong light interfering with the plant's light signal pathway.

[0042] 1. Control group: Add 5 μL of pure water containing 0.05% Tween-20, and record the concentration as 0 μmol / L.

[0043] 2. Treatment Group: Immediately after the seedlings are decapitated, use a pipette to take 5 μL of a 0.1 μmol / L solution of 24-epibranolactone (the preparation process is as follows: prepare a 20 mmol / L stock solution of 24-epibranolactone with anhydrous ethanol, then dilute the stock solution with water to the specified concentration, and add a final concentration of 0.05% Tween-20, referred to as 24e-BL solution), and accurately drop it onto the depression at the seedling growth point, taking care to prevent the solution from dripping onto the cotyledons and hypocotyl; before actual application, a concentration gradient pre-experiment can be set up (see Example 2) to screen the optimal concentration suitable for different watermelon materials.

[0044] S2, Segmented Light Controlled Culture: 1. First stage: Culture in complete darkness at 28℃ for 24 hours to eliminate light interference and fully activate the plant's BR signaling pathway.

[0045] 2. Second stage: 28℃, light intensity 50 μmol·m -2 ·s -1 Cultivating the plants in low light conditions for 12 hours amplifies the differences in BR sensitivity and prevents excessive yellowing of seedlings.

[0046] This embodiment verifies the effectiveness of the method under the optimal parameters of segmented light-controlled cultivation. Those skilled in the art will understand that within the parameter range, by appropriately adjusting the dark time, weak light time, or light intensity for different watermelon varieties, as long as the core principle of "first activating the BR signal in darkness, then amplifying the difference in weak light and maintaining seedling health" is not deviated from, similar identification results can be achieved.

[0047] S3, Comprehensive Scoring Based on Multiple Indicators: After the segmented light-controlled culture was completed, the cotyledon petiole length, true leaf length, and hypocotyl curvature of the seedlings were measured. Based on the measured values ​​or assigned values ​​and the corresponding preset weights, the comprehensive score S was calculated according to Equation 1: S = 0.4 × a + 0.3 × b + 0.3 × c (Equation 1) In Formula 1, a is the measured value of cotyledon petiole length, with the measurement reference being the distance from the growing point to the surface of the cotyledon, in cm; b is the measured value of true leaf length, with the measurement reference being the distance from the growing point to the highest point of the true leaf, in cm; c is the hypocotyl curvature assignment, assigned based on the degree of twisting of the hypocotyl, with a value range of 0 to 1; 0.4, 0.3, and 0.3 are the preset weights of the corresponding indicators.

[0048] Figure 1 This diagram illustrates the methods for measuring the length of cotyledons, true leaves, and hypocotyl curvature in seedlings. Figure 1 (a) Indicates the location for measuring the length of the cotyledon petiole; Figure 1 (b) Indicates the measurement location for the true leaf length; Figure 1 (c) is a schematic diagram of the standard for assigning curvature values ​​to the hypocotyl.

[0049] The hypocotyl curvature c is assigned a value based on the degree of hypocotyl torsion, ranging from 0 to 1. c=0 indicates that the hypocotyl is completely upright, c=1 indicates that the hypocotyl is severely tortuous, and the larger the c value, the higher the degree of bending or torsion of the hypocotyl. Figure 1(c) The hypocotyl morphology at c=0, c=0.2, c=0.5, and c=1 is shown as a reference for assignment. For intermediate states between the above references, values ​​can be assigned linearly within the corresponding range according to the actual degree of torsion. For example, when the degree of curvature is between c=0.2 and c=0.5, a value of 0.3 or 0.4 can be assigned; when it is between c=0.5 and c=1, a value of 0.6, 0.7, 0.8, or 0.9 can be assigned.

[0050] S4. Sensitivity determination: The sensitivity of watermelon seedlings to brassinosteroids is determined by a comprehensive score S. A score S higher than a preset threshold is considered highly sensitive, while a score S lower than the preset threshold is considered low sensitive.

[0051] Example 2 This embodiment focuses on a specific watermelon variety and obtains preset thresholds for effective 24-epibrassinolide treatment concentration and S value.

[0052] The material used for verification was a key regulatory gene for watermelon BR signaling, which was previously obtained by the inventors' team. ClGL1 The two genotypes are near-isogenic lines NIL-1 and NIL-2.

[0053] ClGL1 The gene is numbered Cla97C03G066390 (97103V2.5), and its full-length sequence of the Type-1 genotype is shown in SEQ ID NO:1, while the full-length sequence of the Type-2 genotype is shown in SEQ ID NO:2. NIL-1 carries the Type-1 genotype ( ClGL1 Enhanced function, BR sensitive), NIL-2 carries Type-2 genotype ( ClGL1 (Weak function, weak BR sensitivity).

[0054] Type-2 genotype is watermelon ( Citrullus lanatus One wild-type (WT) gene in ) ClGL1 It is located on chromosome 3, with a physical position of 29790268..29791752 (- chain), and a total length of 1,485 bp.

[0055] Type-1 genotype is ClGL1 A functional variant carried in the Klondike Black Seeded (KBS) cultivar has a full-length genome sequence identical to the wild-type (WT) sequence, with the only difference being the change of nucleotide 597 in exon 3 from G to A (a G→A nonsynonymous point mutation). This mutation results in the change of amino acid 199, encoding the protein, from aspartic acid (Asp) to asparagine (Asn).

[0056] 1. Optimal treatment concentration screening: Using NIL-1 and NIL-2 seedlings as experimental materials, and following the method described in Example 1, six concentration gradients of 24-epibrassinolol (0 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, and 1 μmol / L) were set up, with 15 seedlings treated at each gradient.

[0057] Except for the change in concentration, the other conditions remained the same as in Example 1.

[0058] After treatment, the comprehensive score S value of seedlings in each treatment group was calculated according to Formula 1. The results are expressed as mean ± standard deviation, using Student's score. t The significance analysis was performed, and the results are shown in [the table]. Figure 2 .

[0059] Figure 2 The results showed that within the concentration range of 0.05 μmol / L to 1 μmol / L of 24-epibrassinolide, the differences in the comprehensive score S value between NIL-1 and NIL-2 seedlings were extremely significant (P<0.01). However, at high concentrations, the difference in S value between the two groups narrowed and the distinguishability decreased.

[0060] Specifically, the NIL-1 seedling group ( Figure 2 (Middle blue column) As the concentration of 24-epibrassinolactone increased from 0 μmol / L to 1 μmol / L, the S value continuously increased from about 0.45 to about 2.2, showing a good dose-dependent effect.

[0061] NIL-2 seedling group ( Figure 2 (In the medium gray column) At concentrations of 0.1 μmol / L and below, the S value almost stagnated at 0.4–0.5, showing extremely weak growth. However, when the concentration increased to 0.2 μmol / L and above, the S value of NIL-2 also showed a significant increase, reaching approximately 1.3 at 1 μmol / L. This is mainly because NIL-2 produced a nonspecific stoichiometric saturation response under high concentrations of exogenous hormone stress, weakening its ability to distinguish between sensitivity differences at this concentration.

[0062] Based on the above differences, a preset threshold (definition standard) for the S-value used to distinguish between the two genotype phenotypes was further clarified. According to the distribution of S-values ​​at different concentrations, this threshold increases stepwise with concentration. Considering the stability of the NIL-2 phenotype at low concentrations and the smallest p-value between the two groups (the most statistically significant difference), 0.1 μmol / L was determined to be the optimal treatment concentration.

[0063] 2. Phenotypic identification of the F2 population: Fifty plants (plant numbers 1-50 in Table 1) were obtained from the cross between NIL-1 and NIL-2. Seedlings were cultivated according to the method described in Example 1 (the concentration of 24-epibrassinolide in the 24e-BL solution was 0.1 μmol / L). After the seedlings reached the capping stage, they were treated uniformly. After 36 hours, the cotyledon petiole length, true leaf length and hypocotyl curvature of each individual plant were measured, and the comprehensive score S was calculated according to Formula 1.

[0064] Genomic DNA was extracted from 50 F2 seedlings that underwent phenotypic identification and obtained a comprehensive score of S. ClGL1 PCR detection was performed using specific molecular markers. The results are shown in Table 1.

[0065] Table 1: S-values ​​and genotypes of F2 hybrid seedlings after treatment with brassinolide (24-24) As shown in Table 1, after genotyping, the maximum S value of the 12 Type-2 homozygous (BR weakly sensitive) seedlings was 0.64, while the minimum S value of the 38 seedlings containing the Type-1 genotype (BR sensitive) was 0.77. The S values ​​of all sensitive types were greater than those of the weakly sensitive types.

[0066] According to the method of the present invention, the average value of the maximum S value (0.64) of the weakly sensitive type and the minimum S value (0.77) of the sensitive type is taken to obtain the preset threshold of S value = (0.64+0.77) / 2 = 0.705.

[0067] Example 3 This embodiment is used to verify the ability of the method of the present invention to rapidly screen seedlings of fruit-shaped related lines in actual breeding work.

[0068] The test material was a watermelon germplasm resource, TP103, discovered by our team during breeding work. Its self-pollinated offspring exhibited segregation of fruit shape traits, with the ratio of long-fruited to round-fruited plants conforming to the 3:1 genetic segregation rule controlled by a single gene. However, the target gene regulating fruit shape remains unclear. It should be noted that TP103 is only used as an exemplary material to verify the effectiveness of this method; this method is also applicable to the screening of traits related to BR sensitivity in other watermelon germplasm resources.

[0069] To determine the optimal S-value threshold suitable for this TP103 genetic background, 15 seedlings from the offspring of TP103 with rounder fruit (containing a homozygous recessive target gene, and the offspring do not segregate) and 15 seedlings from the self-pollination offspring of TP103 with elongated fruit (without segregation for three consecutive generations of self-pollination, containing a homozygous dominant target gene) were selected. The optimal treatment concentration of 24-epibrassinolide and the optimal S-value threshold were determined according to the method in Example 2. The results showed that the optimal 24-epibrassinolide treatment concentration for rapid screening of watermelon germplasm resources under this genetic background was 0.2 μmol / L. The S-values ​​of all seedlings with rounder fruit were lower than those of all seedlings with elongated fruit. The average of the maximum S-value (0.735) of the rounder group and the minimum S-value (1.390) of the elongated group was taken, yielding the optimal S-value threshold for this genetic background: (0.735 + 1.390) / 2 = 1.0625.

[0070] To determine the fruit shape index threshold applicable to the TP103 genetic background: among the fruits produced by this material, 10 extreme fruits of the pre-round type and 10 fruits of the pre-long type were selected, and their fruit shape indices were calculated.

[0071] The maximum value of the fruit shape index for rounder fruits is Max. 圆 =1.30, the minimum fruit shape index for elongated fruits is Min. 长 =1.45. Therefore, the preset threshold for the fruit shape index is (1.30 + 1.45) / 2 = 1.375.

[0072] Thus, early screening criteria based on genetic background were established: the S value threshold was set at 1.0625, and the fruit shape index threshold was set at 1.375. That is, seedlings with a comprehensive score S of less than 1.0625 during the seedling stage will have rounder fruits after maturity, and the fruit shape index should not exceed 1.375.

[0073] Verification process: Twenty seedlings (seedling numbers 1-20) of TP103 self-pollinated progeny were selected and subjected to BR sensitivity testing according to the method described in Example 1. The comprehensive score S value of each individual plant was determined.

[0074] After identification, all seedlings were transplanted to the field for normal cultivation and management. After the fruit matured, the longitudinal and transverse diameters of each fruit on each plant were measured uniformly, and the fruit shape index (longitudinal diameter / transverse diameter) was calculated. The results are shown in Table 2. Figure 3 .

[0075] Table 2: S-value of seedlings and fruit shape index of mature fruit from self-pollinated progeny of watermelon germplasm resource TP103 As shown in Table 2, the S-values ​​of the 20 TP103 self-pollinated progeny seedlings exhibited significant differentiation. Using the established threshold S=1.0625 as the boundary, 15 plants (seedling numbers 1–3, 5, 6, 8, 9, 11, 12, 14–16, 18–20) had S-values ​​higher than 1.0625, ranging from 1.376 to 1.751, belonging to the high group; 5 plants (seedling numbers 4, 7, 10, 13, 17) had S-values ​​lower than 1.0625, ranging from 0.696 to 0.756, belonging to the low group. Furthermore, the ratio of high-group to low-group plants was 15:5 = 3:1, consistent with the single-gene segregation pattern of fruit shape traits in this germplasm resource.

[0076] After the fruit ripens (see...) Figure 3 In this study, the 15 plants in the high S-value group all produced elongated fruits with a fruit shape index ranging from 1.40 to 1.75. The 5 plants in the low S-value group (corresponding to seedling numbers 4, 7, 10, 13, and 17) all produced nearly round fruits with a fruit shape index ranging from 1.08 to 1.31. Seedling number 10 had the lowest fruit shape index (1.08), and its fruit was closest to a perfect circle. In this example, the fruit shape indices (1.08–1.31) of the 5 plants in the low-value group did not exceed this threshold, while the fruit shape indices (1.40–1.75) of the 15 plants in the high-value group were all higher than this threshold, verifying the accuracy of the method. It is evident that there is a strong correlation between the S-value at the seedling stage and the fruit shape index at maturity; the fruit shape index of plants with low S-values ​​is significantly lower than that of plants with high S-values.

[0077] Furthermore, the BR-sensitive / weakly sensitive materials obtained by screening using this method can provide basic material support for the discovery and verification of functional genes related to the watermelon BR signaling pathway.

[0078] It should be noted that Examples 2 and 3 demonstrate the universality of the present invention. The watermelon materials NIL-1 and NIL-2 used in Example 2 are targeted at a single known functional gene ( ClGL1 The near-isogenic lines constructed in Example 2 have a highly homogeneous genetic background. The TP103 used in Example 3 is a naturally occurring variant germplasm derived from field breeding, and its genetic background is completely different from that of the material in Example 2. The two groups of materials are unrelated in terms of genomic DNA similarity or germplasm geographical origin, yet the method of this invention accurately distinguished individuals with different BR sensitivity and successfully predicted the fruit shape at maturity. This fully demonstrates that, as long as a specific threshold is determined for the target breeding population, the method of this invention has broad applicability to different watermelon germplasm resources and is not limited to a specific genetic background.

[0079] The embodiments described are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rapid method for identifying the sensitivity of watermelon seedlings to brassinosteroids, characterized in that, Includes the following steps: S1. Seedling treatment: Select watermelon seedlings in the capping stage whose cotyledons have not fully unfolded, and add 5 μL of 0.05 μmol / L to 1 μmol / L 24-epibrassinolide solution to the growing point of the seedling. S2. Segmented controlled light culture: The seedlings treated in step S1 were first cultured in complete darkness for 18-30 hours, and then cultured under low light conditions for 8-16 hours. The light intensity under the low light conditions was 40 μmol·m⁻¹. -2 ·s -1 ~60 μmol·m -2 ·s -1 ; S3. Multi-index comprehensive score: After the segmented light-controlled culture is completed, the cotyledon petiole length, true leaf length and hypocotyl curvature of the seedling are measured. The comprehensive score S is calculated based on the measured values ​​or assigned values ​​and the corresponding preset weights. S4. Sensitivity determination: The sensitivity of watermelon seedlings to brassinosteroids is determined based on the comprehensive score S. A value of S higher than the preset threshold is considered highly sensitive, and a value of S not higher than the preset threshold is considered low sensitive.

2. The method according to claim 1, characterized in that, The watermelon seedlings in step S1 that have reached the "hat removal" stage are seedlings obtained by cultivating them in a completely dark environment at 28℃ for 5 days after sowing.

3. The method according to claim 1, characterized in that, The dropping operation in step S1 is performed in a low-light environment, where the light intensity is 40 μmol·m⁻¹. -2 ·s -1 ~60 μmol·m -2 ·s -1 .

4. The method according to claim 1, characterized in that, The complete darkness culture time in step S2 is 24 hours, the low light culture time is 12 hours, and the light intensity of the low light is 50 μmol·m⁻¹. -2 ·s -1 .

5. The method according to claim 1, characterized in that, The temperature for both the dark culture and the low-light culture was 28°C.

6. The method according to claim 1, characterized in that, In step S3, the cotyledon petiole length is the distance from the growing point to the cotyledon leaf surface; the true leaf length is the distance from the growing point to the highest point of the true leaf; the hypocotyl curvature is assigned a value based on the degree of hypocotyl twisting, with a value range of 0 to 1, where 0 is completely upright and 1 is severely twisted.

7. The method according to claim 1, characterized in that, In step S3, the overall score S is calculated according to formula 1: S = 0.4 × a + 0.3 × b + 0.3 × c (Equation 1) In Formula 1, a is the cotyledon petiole length in cm; b is the true leaf length in cm; c is the hypocotyl curvature value, ranging from 0 to 1; 0.4, 0.3, and 0.3 are the preset weights of the corresponding indicators.

8. The method according to claim 1, characterized in that, The preset threshold in step S4 is determined in the following way: take at least 10 watermelon seedlings of known BR sensitive type and BR weakly sensitive type under the corresponding genetic background, obtain a comprehensive score S according to steps S1 to S3, and when the S value of all sensitive seedlings is greater than the S value of all weakly sensitive seedlings, the average value of the minimum S value among the sensitive seedlings and the maximum S value among the weakly sensitive seedlings is taken as the preset threshold.

9. The application of the method according to any one of claims 1 to 8 in screening watermelon BR signaling pathway mutants and breeding resources with abnormal BR signal sensitivity.

10. The application of the method according to any one of claims 1 to 8 in early screening of watermelon fruit shape, characterized in that, Watermelon seedlings with a seedling comprehensive score S lower than the preset threshold under the same genetic background will have fruits with a rounder shape after maturity, and their fruit shape index will not exceed the fruit shape index threshold. The fruit shape index threshold is determined in the following way: Take at least 10 watermelon fruits with relatively round and long shapes under the corresponding genetic background, measure the longitudinal and transverse diameters of each fruit, and calculate the fruit shape index SX = longitudinal diameter / transverse diameter. The average of the maximum value of SX for round fruits and the minimum value of SX for long fruits is used as the fruit shape index threshold.

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