Breeding method for cultivating new heat-resistant bitter gourd variety

Abstract

The invention discloses a breeding method for cultivating a new heat-resistant balsam pear variety, which comprises the following steps: S1, creating a grafting bridge: grafting a balsam pear scion to a heat-resistant related plant stock to obtain a grafted plant; through the interaction of the stock and the scion, the physiological property or epigenetic signal of the stock may influence the pollen development of the scion, so that the scion pollen potentially carries the heat resistance related character of the stock; collecting pollen of the bitter gourd scions on the grafted plants, and granting the pollen to stigmas of the non-grafted bitter gourd maternal plants to obtain hybrid seeds; s2, screening candidate bridge hybrid plants: planting the hybrid seeds, applying high-temperature stress in a seedling stage or a growth stage, and screening out plants with heat resistance remarkably superior to that of a control group as the candidate bridge hybrid plants. The method has the advantages that the genital isolation of species can be effectively overcome, the genetic basis of the bitter gourd is remarkably broadened, and the extreme heat-resistant gene in related wild species of cucurbitaceae can be successfully introduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic breeding and biotechnology, in particular to a breeding method for breeding a new heat-resistant balsam pear variety. BACKGROUND

[0002] Balsam pear is an important vegetable crop widely planted in China, but its growth and development are extremely sensitive to temperature. In the summer high-temperature season, when the environmental temperature is continuously higher than 35℃, balsam pear plants often appear photosynthesis inhibition, pollen abortion, and pollination and fertilization process obstruction, etc., which directly leads to serious flower and fruit drop, high fruit malformation rate, and significant yield and quality decline. Therefore, breeding a new balsam pear variety with stable strong heat resistance is of great significance to guarantee the market supply in summer and autumn off-season and stabilize the income of vegetable farmers.

[0003] At present, the genetic improvement and new variety breeding of balsam pear mainly rely on intraspecific hybridization and backcross breeding, etc. In the patent literature with Chinese patent number CN108651268B, a breeding method for a balsam pear gynoecious line is disclosed, which is based on chemical induced male and backcross technology for sex type transformation. Although this method is efficient, its genetic operation is completely limited within the balsam pear species. Another patent literature with Chinese patent number CN114854901A discloses a molecular marker co-segregated with the balsam pear gynoecious trait and its application. Although this technology reflects the advancement of molecular marker assisted selection, the object of screening is still the genetic variation within the balsam pear species. Therefore, the existing technical solutions generally have a common limitation: the gene resources used are limited within the cultivated balsam pear species, and the genetic basis is relatively narrow. This leads to the fact that the existing breeding methods have difficulty in making breakthrough progress in the improvement of complex traits such as heat resistance of balsam pear, which is controlled by multiple genes, and the breeding effect has encountered a bottleneck.

[0004] In summary, the fundamental problem of the existing technical system in the improvement of balsam pear heat resistance is the lack of technical means that can effectively broaden its genetic basis and utilize excellent gene resources in distant species. Specifically, the existing technologies do not provide a feasible solution that can overcome the reproductive isolation barrier of species, successfully introduce the extremely heat-resistant genes in the wild relatives of the Cucurbitaceae family into the cultivated balsam pear, and ultimately create new germplasm with breakthrough heat-resistant traits. SUMMARY

[0005] The present application relates to the field of plant genetic breeding and biotechnology, in particular to a breeding method for breeding a new heat-resistant balsam pear variety.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The application discloses a breeding method for cultivating a new heat-resistant balsam pear variety, and belongs to the technical field of plant breeding. S1, grafting bridge creation: grafting balsam pear scions onto heat-resistant relative plant rootstocks to obtain grafted plants; through the interaction between the rootstock and the scion, the physiological characteristics or epigenetic signals of the rootstock can affect the pollen development of the scion, so that the pollen of the scion potentially carries heat-resistant related traits of the rootstock; collecting the pollen of the balsam pear scion on the grafted plant, and pollinating the stigma of an ungrafted balsam pear female parent plant to obtain hybrid seeds; S2, candidate bridge hybrid plant screening: planting the hybrid seeds, applying high-temperature stress at the seedling stage or the growth stage, and screening plants with significantly better heat resistance than a control to serve as candidate bridge hybrid plants; S3, backcross breeding: taking the candidate bridge hybrid plant as a heat-resistant donor parent and a balsam pear cultivar with excellent comprehensive traits as a recurrent parent, performing multi-generation backcross, applying high-temperature stress to each generation of backcross offspring and screening heat-resistant single plants; S4, self-pollination purification: self-pollinating the backcross offspring, stabilizing the heat-resistant traits and other agronomic traits under selection pressure, and obtaining a heat-resistant balsam pear new variety with genetic stability.

[0007] Preferably, in step S1, the heat-resistant relative plant rootstock is a wild heat-resistant species of the Cucurbita genus or the Cucurbita genus.

[0008] By means of artificial detasseling and bagging isolation, the application effectively avoids self-pollination and contamination of external pollen, ensures the purity and genetic consistency of the hybrid seeds, significantly improves the success rate and reliability of distant hybridization, reduces experimental errors caused by non-target pollination, provides high-quality genetic materials for subsequent screening, enhances the controllability and repeatability of the breeding process, accelerates the introduction and stabilization of heat-resistant traits, and thus improves the overall breeding efficiency and lays a solid foundation for cultivating a new balsam pear variety with excellent heat resistance.

[0009] Preferably, in step S1, the female flowers of the ungrafted balsam pear female parent plant are artificially detasseled and bagged before pollination.

[0010] By setting high-temperature stress conditions of 35-40 DEG C during the day and 28-30 DEG C at night, the application simulates a natural high-temperature environment, effectively induces the expression of heat-resistant phenotypes of the plants, and facilitates the accurate screening of individuals with real heat resistance. The advantage is that it improves the accuracy and efficiency of screening, avoids false positive or false negative results caused by excessively low or high temperatures, ensures that the selected plants perform stably in actual production environments, and thus accelerates the development and application of heat-resistant balsam pear varieties.

[0011] Preferably, in step S2, the high-temperature stress conditions are: daytime temperature 35-40℃, nighttime temperature 28-30℃.

[0012] By comprehensively evaluating multiple indicators such as leaf wilting index, photosynthetic efficiency, fruit setting rate, and fruit malformation rate, the application achieves comprehensive and objective evaluation of heat tolerance. This multi-index method overcomes the limitations of single indicators and can more accurately reflect the overall physiological state and agronomic performance of plants under high temperature. It improves the reliability and scientificity of the screening, reduces the risk of misselection, and ensures that the selected varieties not only have strong heat tolerance but also have excellent production traits, thereby enhancing the market adaptability and promotion value of the new bitter gourd variety.

[0013] Preferably, in step S2, the evaluation index of heat tolerance includes one or more of leaf wilting index, photosynthetic efficiency, fruit setting rate, and fruit malformation rate.

[0014] By using molecular marker technology for genotyping, the application can quickly confirm that the candidate bridge hybrid plants contain specific nucleic acid fragments from the rootstock plants, thereby verifying the genetic source of heat tolerance traits. This method improves the accuracy and efficiency of screening and avoids errors that may arise from relying solely on phenotypes. It enables early genotypic selection, accelerates the breeding process, and ensures the stable introduction of heat tolerance genes, providing a reliable genetic foundation for subsequent backcrossing and selfing steps, ultimately promoting the rapid cultivation and industrialization of heat-tolerant bitter gourd new lines.

[0015] Preferably, in step S2, after screening the candidate bridge hybrid plants with excellent phenotypes, further identification is performed using physiological and biochemical indicators or molecular marker technology to confirm that the heat tolerance traits are associated with the rootstock plants.

[0016] Preferably, in step S3, the multiple generations of backcrossing are 3-4 generations.

[0017] By using molecular markers linked to heat tolerance major genes for early assisted selection of backcross progeny, the application achieves efficient screening of single plants carrying target heat tolerance genes, significantly improving the accuracy and efficiency of the backcrossing process. This method avoids the lag and uncertainty of traditional phenotypic screening, can quickly identify excellent individuals in early generations, reduces unnecessary backcross generations and resource waste, accelerates the transfer and fixation of heat tolerance traits, while maintaining the excellent agronomic traits of the recurrent parent, shortens the breeding cycle, and provides technical support for breeding heat-tolerant bitter gourd varieties with excellent comprehensive traits.

[0018] Preferably, during the backcrossing process in step S3, molecular markers linked to heat tolerance major genes are used for early assisted selection of backcross progeny to screen single plants carrying target heat tolerance genes for the next round of backcrossing.

[0019] Through selfing of the backcross progeny for 2-3 generations, and combining field phenotypic identification and laboratory physiological index detection, the application effectively stabilizes the heat tolerance and other agronomic traits, and eliminates the trait separation single plant. This step ensures the genetic purity and trait consistency of the new strain, improves the stability and generalizability of the variety, and through multiple generations of selection and stress testing, it strengthens the expression of heat tolerance, optimizes the agronomic performance, reduces the risk of variation in subsequent production, and lays a solid foundation for large-scale application of the heat-tolerant balsam pear new variety.

[0020] Preferably, in step S4, the selfing is for 2-3 generations, and after each generation of selfing, field phenotypic identification and laboratory physiological index detection are performed to eliminate trait separation single plants and retain single plants with stable heat tolerance and excellent agronomic traits.

[0021] By setting the original balsam pear female parent plants that have not been subjected to distant hybridization and are grown under the same sowing time, water and fertilizer, and cultivation management conditions as the candidate bridge hybrid plants as controls, the application ensures the fairness and comparability of heat tolerance screening, which improves the reliability of the screening results, facilitates accurate identification of individuals with true heat tolerance advantages, accelerates the breeding process of excellent varieties, and at the same time provides a reliable reference benchmark for subsequent breeding work.

[0022] Preferably, in step S2, the control is the original balsam pear female parent plant that has not been subjected to distant hybridization and is grown under the same sowing time, water and fertilizer, and cultivation management conditions as the candidate bridge hybrid plant.

[0023] By setting the original balsam pear female parent plants that have not been subjected to distant hybridization and are grown under the same sowing time, water and fertilizer, and cultivation management conditions as the candidate bridge hybrid plants as controls, the application ensures the fairness and comparability of heat tolerance screening, which eliminates the influence of environmental and management factors on the experimental results, making the heat tolerance evaluation more objective and accurate.

[0024] Compared with the prior art, the application has the following beneficial effects: 1、The application overcomes the reproductive isolation barrier between balsam pear and wild species of the Cucurbitaceae family (such as the genus Ecballium and the genus Cucurbita) by creating the key step of "grafting a bridge", specifically by grafting balsam pear scions onto wild heat-tolerant rootstocks, and then using the pollen of the grafted plants to backcross with balsam pear female parents, successfully realizing the penetration of distant genes, and for the first time introducing the extremely heat-tolerant traits from wild species into cultivated balsam pear, fundamentally solving the heat tolerance improvement bottleneck problem caused by the narrow genetic basis in the prior art; 2、The present application ensures the accuracy and efficiency of the introduction and screening of heat tolerance traits by constructing a double screening system integrating "high temperature stress phenotype screening" and "molecular marker genotyping". By applying specific high temperature stress (35-40℃ during the day / 28-30℃ at night) at seedling stage and growth stage and comprehensively evaluating physiological and agronomic indicators such as leaf wilting index and fruit setting rate, individuals with excellent phenotype can be accurately screened out, and further molecular marker technology is used to confirm that they contain specific nucleic acid fragments from the rootstock, realizing the tracking of target genotypes in early generations, significantly improving the breeding efficiency and reliability. 3、The present application successfully integrates and fixes the exogenous heat tolerance gene and excellent agronomic traits by combining the breeding program of "multi-generation backcross transformation" and "self-purification", uses the selected candidate bridge hybrid plants as donors, and uses excellent cultivated species as recurrent parents for multiple rounds of backcross, and continuously applies selection pressure during backcrossing and selfing, effectively eliminating the undesirable traits accompanied by wild parents, quickly restoring the comprehensive traits of recurrent parents, and finally obtaining a new bitter gourd line with stable genetics, strong heat tolerance and excellent agronomic traits, realizing the creation of a breakthrough germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 The overall flow of the breeding method and the flowchart of the bridge hybrid creation of the present application are shown; Fig. 2 The backcross transformation flowchart of the present application is shown; Fig. 3 The self-purification flowchart of the present application is shown. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] In order to solve the problem of narrow genetic basis in the breeding of heat-tolerant bitter gourd in the prior art, the following technical solutions are given, please refer to Figs. 1-3 ; A breeding method for breeding heat-tolerant new varieties of bitter gourd, comprising the following steps: S1, grafting bridge creation: grafting the bitter gourd scion to a heat-tolerant relative plant stock to obtain a grafted plant; through the interaction between the stock and the scion, the physiological characteristics or epigenetic signals of the stock can affect the pollen development of the scion, so that the pollen of the scion potentially carries the heat tolerance-related traits of the stock; collecting the pollen of the bitter gourd scion on the grafted plant and pollinating the stigma of an ungrafted bitter gourd female parent plant to obtain hybrid seeds; S2, candidate bridge hybrid plant screening: planting the hybrid seeds and applying high temperature stress at the seedling stage or growth stage to screen out plants with significantly better heat tolerance than the control as candidate bridge hybrid plants; S3, backcross breeding: using the candidate bridge hybrid plant as the heat tolerance donor parent and a bitter gourd cultivar with excellent comprehensive traits as the recurrent parent to perform multiple generations of backcross, and applying high temperature stress and screening heat-tolerant single plants in each generation of backcross offspring; S4, self-purification: selfing the backcross offspring to stabilize the heat tolerance traits and other agronomic traits under selection pressure to obtain a genetically stable heat-tolerant bitter gourd new line.

[0028] Preferably, in step S1, the heat-tolerant relative plant stock is a wild heat-tolerant species of the genus Cucurbita or Cucurbita in the Cucurbitaceae family.

[0029] Preferably, in step S1, the female flowers of the ungrafted bitter gourd female parent plant are artificially emasculated and bagged before pollination.

[0030] Preferably, in step S2, the high temperature stress conditions are: daytime temperature 35-40℃, nighttime temperature 28-30℃.

[0031] Preferably, in step S2, the evaluation index of heat tolerance includes one or more of leaf wilting index, photosynthetic efficiency, fruit setting rate, and fruit malformation rate.

[0032] Preferably, in step S2, after screening out the candidate bridge hybrid plant with excellent phenotype, further physiological and biochemical index detection or molecular marker technology is used for identification to confirm that the heat tolerance traits of the plant are associated with the stock plant.

[0033] Preferably, in step S3, the multiple generations of backcross are 3-4 generations.

[0034] Preferably, during the backcrossing process in step S3, molecular markers linked to heat tolerance major genes are used for early assisted selection of the backcross offspring to screen out single plants carrying the target heat tolerance gene for the next round of backcrossing.

[0035] Preferably, in step S4, the selfing is 2-3 generations, and after each generation of selfing, the single plants with separated traits are eliminated and the single plants with stable heat tolerance and excellent agronomic traits are retained through field phenotype identification and laboratory physiological index detection.

[0036] Preferably, in step S2, the control is a wild momordica cochinchinensis plant that is not subjected to wide cross breeding and is grown under the same water and fertilizer and cultivation management conditions as the candidate bridge hybrid plant.

[0037] Example 1: Using wild momordica cochinchinensis to create a new heat-resistant balsam pear line "Reyu No. 1" 1. Material preparation Rootstock: Select the seeds of the extremely heat-resistant wild momordica cochinchinensis, which can still grow normally at 40°C after high-temperature domestication and screening; Scion: Select the commercial balsam pear cultivar "Cuiyu" which has excellent fruit shape and moderate bitterness but weak heat resistance; Female parent: "Cuiyu" balsam pear pure line plant of the same origin as the scion, to ensure genetic background consistency.

[0038] 2. Grafting bridge creation (S1) Top grafting method is used for grafting: when the wild momordica cochinchinensis rootstock seedlings grow to 3 leaves 1 heart, stem diameter 0.3-0.5 cm, the top growth point is cut off, and a 1 cm deep longitudinal cut is made in the center of the stem; Select "Cuiyu" balsam pear scion seedlings (2 leaves 1 heart), cut into a wedge shape and insert into the cut of the rootstock, and fix it with a grafting clamp. Place it in a humidity of 85% and a temperature of 25-28°C for 10 days to obtain a surviving grafted plant; Artificial emasculation and bagging isolation: before the balsam pear scion flowers of the grafted plant bloom, artificially emasculate the healthy female flowers of the "Cuiyu" female parent plant (remove the stamens to avoid self-pollination), and immediately bag them with breathable sulfuric acid paper bags, and mark the emasculation date; Cross-pollination: Collect fully opened balsam pear scion flowers from the grafted plant, evenly apply pollen to the stigma of the female parent plant, re-bag after pollination and mark the cross combination. After the fruit is fully mature, collect the seeds, which are F1 generation bridge hybrid seeds.

[0039] 3. Candidate bridge hybrid plant screening (S2) Sow F1 generation seeds and grow them conventionally to 3-4 true leaf stage, then transplant them to an artificial climate room and set up high temperature stress conditions: daytime temperature 38°C, nighttime temperature 29°C, stress for 7 days, during which maintain light for 12h / d and humidity 70%; Control setting: Use "Cuiyu" balsam pear plants grown under the same water and fertilizer and cultivation management conditions as the candidate bridge hybrid plant as the control; Phenotypic screening: Screen based on leaf wilting index and fruit setting rate. After stress, the control plant leaf wilting index reached 78.4±5.1%, and the fruit setting rate was only 28.3±3.9%. 10 plants with leaf wilting index <30% and stable growth trend were selected; Genotype identification: Genomic DNA of 10 candidate plants was extracted, and specific SCAR molecular markers of C. colocynthis were used for PCR amplification. The results showed that 3 plants could amplify target specific bands, confirming that they contained specific nucleic acid fragments from wild C. colocynthis, and were defined as candidate bridge hybrid plants.

[0040] 4. Backcross breeding (S3) The 3 candidate bridge hybrid plants were used as heat-tolerant donor parents, and the C. colocynthis cultivar “Cuiyu” was used as the recurrent parent for the first backcross, obtaining the BC1F1 population. After each generation of backcross, seedlings (3-4 true leaf stage) were subjected to high temperature stress with 35-40°C during the day and 28-30°C at night, and early assisted selection was performed using KASP molecular markers linked to the heat-tolerant major gene to screen single plants that not only exhibited heat-tolerant phenotype (leaf wilting index <40%) but also carried the target heat-tolerant gene for the next round of backcross. A total of 3 generations of backcross were performed. By the BC3F1 generation, the genetic background recovery rate reached more than 93% through molecular marker detection, and the heat-tolerant traits were stable, with field comprehensive traits (plant type, leaf shape, flowering period) being basically consistent with those of the recurrent parent “Cuiyu”.

[0041] 5. Self-pollination purification (S4) BC3F1 elite single plants were selected for self-pollination, obtaining the BC3F2 population. Each generation of self-pollinated offspring was planted under high temperature stress environment (natural summer open field, daytime temperature ≥ 35°C), and through field phenotype identification (leaf wilting index, fruit setting rate) and laboratory physiological index detection (photosynthetic efficiency), unstable trait single plants were eliminated, and single plants with stable heat tolerance and excellent fruit commodity quality were retained. After 2 generations of continuous self-pollination to the BC3F4 generation, the heat tolerance and agronomic traits of the offspring plants were not separated, and a genetically stable heat-tolerant balsam pear new strain was obtained, named “Reyu No. 1”.

[0042] Example 2: Creation of heat-tolerant balsam pear new strain “Xiaofeng No. 1” using wild pumpkin 1. Material preparation Stock: A wild heat-tolerant species of Cucurbitaceae from tropical regions was selected, with a heat tolerance threshold of 42°C, and was identified to contain 3 heat-tolerant major QTL loci. Scion and female parent: The early-maturing, high-yielding, and green balsam pear cultivar “Lubao” was selected. This variety has high market recognition, but the fruit setting rate under high summer temperature is only about 25%.

[0043] 2. Grafting bridge creation (S1) Grafting: Wild pumpkin rootstock and "Green Bao" bitter gourd scion (both 2 leaf 1 heart seedlings) were cut at a 30° angle, with a cut length of 0.8-1 cm. After aligning the cambium, a grafting film was wrapped and fixed. During the seedling recovery period, the temperature in the nursery was maintained at 26-29°C and the humidity was 90%. After 15 days, the grafting film was removed, and the grafted plants were obtained. Artificial emasculation and crossing: The female flowers of the "Green Bao" female parent were emasculated one day in advance and bagged. When the scion male flowers of the grafted plants opened, pollen was collected for pollination. After pollination, the plants were bagged and labeled. The F1 bridge hybrid seeds were harvested after maturity.

[0044] 3. Screening of candidate bridge hybrid plants (S2) The F1 bridge hybrid seeds were sown and grown in the open field until the flowering and fruit setting stage. The plants were subjected to high temperature stress in the field during the natural high temperature season (July-August, with a maximum temperature of >35°C during the day). Control: "Green Bao" bitter gourd seeds were sown at the same time, and the cultivation and management conditions were consistent. Phenotypic screening: The photosynthetic efficiency (Pn value) and fruit setting rate were used as core evaluation indicators. The photosynthetic efficiency of the control plants was only 8.5 μmol・m -2 ・s -1 , and the fruit setting rate was 25%. Eight excellent single plants with photosynthetic efficiency ≥15 μmol・m -2 ・s -1 and fruit setting rate ≥55% were selected. Genotype identification: The candidate single plants were identified using the genomic in situ hybridization (GISH) technique. Specific chromosome fragments of wild pumpkin were observed on the chromosomes of root tip cells during metaphase, confirming that 6 plants were true candidate bridge hybrid plants.

[0045] 4. Backcross breeding (S3) The confirmed candidate bridge hybrid plants were used as donor parents, and "Green Bao" bitter gourd was used as the recurrent parent for backcross breeding. A total of 4 generations of backcross breeding (BC1F1-BC4F1) were conducted. After each generation of backcross breeding, the seedlings were subjected to assisted selection using SSR molecular markers tightly linked to the heat tolerance major QTL to quickly select single plants carrying the target heat tolerance gene. Phenotypic observation under high temperature stress (leaf wilting) was also conducted to ensure the heat tolerance of the backcross progeny. By the BC4F1 generation, the genetic background of the plants was similar to that of the recurrent parent "Green Bao" with a similarity of 95%, and the heat tolerance was significantly better than that of the original variety.

[0046] 5. Self-purification (S4) The BC4F1 excellent single plants were self-pollinated, and continuous self-pollination was conducted for 3 generations (BC4F1-BC4F3). Each generation of self-crossing is screened in high-temperature environment, focusing on fruit setting rate, fruit malformation rate and fruit commodity property, and single plants with malformation rate >10% and irregular fruit shape are eliminated; Finally, a new heat-resistant balsam pear line with genetic stability, fruit setting rate of more than 60% under high temperature and fruit malformation rate of less than 5% is obtained, which is named "Xiaofeng No.1"; Example 3: Utilizing wild balsam pear variety to create heat-resistant balsam pear new line "Shu Kang No.2" 1. Material preparation Stock: Wild balsam pear variety with strong desert adaptability and strong extreme heat resistance, which can tolerate short-term high temperature of 45℃; Scion: High-quality large-fruited balsam pear cultivar "Changbai Balsam Pear", with fruit length of 30-35 cm, but easy to drop flowers and fruits under high temperature; Female parent: "Changbai Balsam Pear" pure line plant.

[0047] 2. Grafting bridge creation (S1) Top grafting: Wild balsam pear stock is topped when it has 4 leaves and 1 heart, and scion is selected as "Changbai Balsam Pear" 2-leaf 1-heart seedling, which is inserted into the stock after being sharpened, and then placed in a shading nursery shed after being fixed. After 12 days of seedling recovery, the survival rate reached 89%; Artificial emasculation and bagging: The female flowers of the female parent are emasculated and bagged one day before opening; the pollen of the scion of the grafted plant is collected to pollinate F1 generation bridge hybrid seeds.

[0048] 3. Candidate bridge hybrid plant screening (S2) Artificial climate chamber high temperature stress: F1 generation seedlings at 3-leaf 1-heart stage, setting day temperature of 40℃ and night temperature of 30℃, lasting for 10 days; Control: Non-hybrid "Changbai Balsam Pear", treated at the same time; Screening index: Leaf wilting index and fruit malformation rate, control wilting index 82%, malformation rate 23%, 5 plants with wilting index <28% and no obvious malformation are screened out; Molecular identification: 4 plants amplified target bands by using balsam pear specific SSR marker detection, which are confirmed as candidate bridge hybrid plants.

[0049] 4. Backcrossing (S3) Using candidate bridge hybrid plants as donors and "Changbai Balsam Pear" as recurrent parent, backcrossing for 3 generations; Each generation is screened by KASP molecular marker assisted selection + high temperature stress phenotype, and single plants with heat-resistant genes and excellent agronomic traits are retained.

[0050] 5. Self-crossing purification (S4) Self-crossing of backcrossing progeny for 2 generations, screening under high temperature, focusing on maintaining large fruit characteristics of "Changbai Balsam Pear" and stabilizing heat resistance; Finally, a genetically stable heat-resistant large fruit type balsam pear new strain "Shu Kang 2" was obtained, and the single fruit weight under high temperature can still reach more than 300 g, and the fruit setting rate is 62%.

[0051] Test example 1: comparison test of heat resistance and comprehensive agronomic traits of "Re Yu 1" 1. Test purpose Verify the heat resistance, yield and fruit quality of "Re Yu 1" under high temperature environment, and analyze the differences with the control variety.

[0052] 2. Materials and methods Test material: Test variety: "Re Yu 1" developed by the application.

[0053] Control variety: Recurrent parent "Cui Yu", local main variety "Xia Shuai".

[0054] Test design: Test site: plastic greenhouse (natural high temperature in summer + artificial control), soil is sandy soil, and fertility is medium.

[0055] Planting method: randomized block design, 3 times of repetition, plot area 15 m 2 , plant spacing 0.5 m x 1.5 m, 20 plants per plot.

[0056] Cultivation management: the water and fertilizer, disease and pest control measures of each variety are consistent, and unified frame cultivation is adopted.

[0057] High temperature stress treatment: During the test period (July-August), through greenhouse film covering + shading net control, the daytime temperature in the greenhouse is maintained at 35-40℃, and the night temperature is 28-30℃, which lasts for 40 days.

[0058] Measurement index and method: a. Leaf wilting index: on the 5th day of continuous high temperature at 14:00, using 5-level grading method (0 level: no wilting; 1 level: ≤25% leaf wilting; 2 level: 26%-50% leaf wilting; 3 level: 51%-75% leaf wilting; 4 level: >75% leaf wilting), calculate the wilting index = [Σ (number of each level x level number) / (total number of plants x highest level number)] x 100%.

[0059] b. Net photosynthetic rate (Pn): 9:00-11:00 in full bloom period, the net photosynthetic rate of functional leaves is determined by portable photosynthetic instrument, 5 plants are determined in each plot, and 3 leaves are determined for each plant.

[0060] c. Fruit setting rate: the effective fruit setting number (fruit transverse diameter ≥3 cm) within 20 days after full bloom is counted, and the fruit setting rate = (effective fruit setting number / flowering number) x 100%.

[0061] d. Single plant yield: Total weight of mature fruits per plant was counted at harvest.

[0062] e. Fruit quality: Fruit length and transverse diameter were measured, and fruit malformation rate = (number of malformed fruits / total number of fruits) x 100% was calculated.

[0063] 3. Test results

[0064] 4. Conclusion Under high temperature stress, the leaf wilting index of "Reyue No. 1" was significantly lower than that of the control variety, the net photosynthetic rate, fruit setting rate and single plant yield were significantly higher than those of the control, the fruit malformation rate was significantly lower than that of the recurrent parent "Cuiyu", and the fruit size had no significant difference with the control, indicating that it not only retains the excellent commodity traits of "Cuiyu", but also has outstanding heat tolerance, which can effectively solve the problem of bitter gourd yield reduction and quality decline caused by high temperature in summer.

[0065] Test example 2: Multi-point regional heat tolerance verification test of "Xiaofeng No. 1" 1. Test purpose Under different ecological regions of high temperature environment, the heat tolerance stability and adaptability of "Xiaofeng No. 1" were verified.

[0066] 2. Materials and methods Test material: Test variety "Xiaofeng No. 1", control variety is local main planting heat-resistant bitter gourd "Yanxiu".

[0067] Test site: 3 test points (A point in South China, B point in East China, C point in Southwest China), all selected summer high temperature frequent plots.

[0068] Test design: Randomized block design, 3 times of repetition, plot area 20 m 2 , plant spacing 0.6 m x 1.4 m, conventional cultivation management.

[0069] High temperature condition: During the test period (June-August) of each test point, the daytime average temperature was 36-39℃, and the nighttime average temperature was 27-30℃.

[0070] Measurement index: Leaf wilting index, fruit setting rate, single plant yield, fruit malformation rate (measurement method same as test example 1).

[0071] 3. Test results

[0072]

[0073] 4. Conclusion "Xiaofeng No. 1" in three different ecological regions of high temperature environment, all show low leaf wilting index, high fruit setting rate, high yield and low fruit malformation rate, significantly better than the control variety "Yanxiu", indicating that its heat tolerance stability, wide adaptability, can be planted in many regions of China in summer high temperature environment.

[0074] Test example 3: "Shikang No. 2" and conventional varieties of high temperature physiological characteristics comparison test 1. Test purpose To verify the heat tolerance mechanism of "Shikang No. 2" from the physiological level, and compare the differences in high temperature adaptability of conventional varieties.

[0075] 2. Materials and methods Test materials: "Shikang No. 2", conventional variety "Changbai bitter gourd" (original scion variety).

[0076] Test design: artificial climate chamber culture, seedling 3 leaves 1 heart stage high temperature stress (daytime 38℃, nighttime 29℃), physiological indexes were measured at 0 day, 3 days, 7 days of stress, respectively.

[0077] Measurement index: a. Malondialdehyde (MDA) content: determination by thiobarbituric acid method, reflecting the degree of cell membrane damage.

[0078] b. Superoxide dismutase (SOD) activity: determination by nitro blue tetrazolium method, reflecting antioxidant capacity.

[0079] c. Chlorophyll content: determination by acetone extraction method, reflecting the stability of photosynthetic apparatus.

[0080] 3. Test results

[0081] 4. Conclusion Under high temperature stress, the MDA content of "Shikang No. 2" was significantly lower than that of "Changbai bitter gourd", indicating that its cell membrane damage was lighter; SOD activity was significantly higher than that of "Changbai bitter gourd", and the antioxidant capacity was stronger; The decline range of chlorophyll content was significantly smaller than that of "Changbai bitter gourd", and the stability of photosynthetic apparatus was better. The above physiological characteristics prove that "Shikang No. 2" realizes stable growth in high temperature environment by enhancing antioxidant capacity, protecting cell membrane and photosynthetic apparatus, and the heat tolerance mechanism is clear.

[0082] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present application will be limited to the appended claims. It must be noted that, as used in the specification and the appended claims, the singular form "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" can include a plurality of components. Similarly, the words "comprise," "comprises," and "comprising," as well as the words "include," "includes," and "including," when used in this specification and in the following claims, are intended to specify the presence of stated features, regions, integers, steps, operations, elements, or components, but they do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, or groups thereof. Furthermore, these terms do not necessarily denote the presence of anything that can be claimed as new. The meaning of "a," "an," and "the" also includes plural references and plural forms, for example, "a" or "an" entity includes one or more entities.

[0083] While the embodiments of the application have been shown and described herein, it is understood that modifications, substitutions, changes, and alterations can be made by those skilled in the art without departing from the spirit and scope of the present application.

Claims

1. A method for breeding new heat-resistant bitter gourd varieties, characterized in that, Includes the following steps: S1. Grafting bridge creation: Bitter gourd scions are grafted onto heat-resistant rootstocks to obtain grafted plants; through the interaction between the rootstock and scion, the physiological characteristics or epigenetic signals of the rootstock may affect the pollen development of the scion, so that the scion pollen may potentially carry the heat-resistant related traits of the rootstock. Pollen from the bitter gourd scion on the grafted plant is collected and transferred to the stigma of the ungrafted bitter gourd mother plant to obtain hybrid seeds. S2. Screening of candidate bridge hybrid plants: Plant the hybrid seeds and apply high temperature stress during the seedling or growth period. Select plants with significantly better heat resistance than the control as candidate bridge hybrid plants. S3. Backcrossing and breeding: Using the candidate bridge hybrid plants as heat-resistant donor parents and bitter gourd cultivars with excellent comprehensive traits as recurrent parents, multiple generations of backcrossing were carried out. High temperature stress was applied to each generation of backcross offspring and heat-resistant single plants were screened. S4. Self-pollination purification: Self-pollinate the backcross offspring to stabilize heat resistance and other agronomic traits under selection pressure, thereby obtaining a genetically stable heat-resistant bitter gourd new line.

2. The breeding method according to claim 1, characterized in that, In step S1, the heat-resistant rootstock is a wild heat-resistant species of Cucurbita or Cucurbita genus in the Cucurbitaceae family.

3. The breeding method according to claim 1, characterized in that, In step S1, before pollination, the female flowers of the ungrafted bitter gourd mother plant are artificially emasculated and bagged for isolation.

4. The breeding method according to claim 1, characterized in that, In step S2, the conditions for high-temperature stress are: daytime temperature 35-40℃ and nighttime temperature 28-30℃.

5. The breeding method according to claim 1, characterized in that, In step S2, the evaluation indicators of heat resistance include one or more of the following: leaf wilting index, photosynthetic efficiency, fruit setting rate, and fruit deformity rate.

6. The breeding method according to claim 1, characterized in that, In step S2, after screening out candidate bridge hybrids with excellent phenotypes, they are further identified using physiological and biochemical index detection or molecular marker technology to confirm that their heat resistance traits are associated with the rootstock plant.

7. The breeding method according to claim 1, characterized in that, In step S3, the multi-generation backcrossing is 3-4 generations.

8. The breeding method according to claim 1 or 7, characterized in that, In the backcrossing process of step S3, molecular markers linked to the major heat-resistant gene are used to perform early auxiliary selection on the backcross progeny in order to screen out individual plants carrying the target heat-resistant gene for the next round of backcrossing.

9. The breeding method according to claim 1, characterized in that, In step S4, the self-pollination is carried out for 2-3 generations. After each generation of self-pollination, field phenotypic identification and laboratory physiological index testing are conducted to eliminate individual plants with segregating traits and retain individual plants with stable heat resistance and excellent agronomic traits.

10. The breeding method according to claim 1 or 5, characterized in that, In step S2, the control is the original bitter gourd mother plant that was not subjected to distant hybridization and was grown under the same water, fertilizer and cultivation management conditions as the candidate bridge hybrid plant.

Images