Passion fruit heat resistance identification method

Abstract

The invention discloses a passion fruit heat resistance identification method, and belongs to the technical field of plant variety identification. The method comprises the following technical steps: (1) heat treatment; (2) index detection; (3) calculating a relative heat resistance index; (4) sequencing heat resistance; (5) grading heat resistance; and (6) heat resistance grade evaluation. According to the method, the heat resistance of the passion fruit can be identified more accurately through high-temperature treatment and normal-temperature contrast treatment and by combining measurement of various physiological and growth indexes (relative conductivity, soluble protein content, soluble sugar content, antioxidant enzyme activity, pollen germination rate, fruit bearing number of a single plant and fruit setting rate), and the relative heat resistance index S value is calculated according to the indexes, so that the heat resistance of the passion fruit can be identified more accurately. The heat resistance of the varieties is sequenced, evaluated and graded according to the relative heat resistance index S value, the heat resistance of the passion fruit varieties or germplasm resources can be evaluated objectively and accurately, and breeding of new heat-resistant varieties and popularization and application of dominant heat-resistant varieties are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of plant variety identification, and particularly relates to a method for identifying the heat resistance of passion fruit. Background Art

[0002] Passion fruit is an important tropical and subtropical fruit tree in southern my country. Due to its unique fragrance, it has the advantages of fresh eating and processing, short planting cycle and fast profit generation. Since Guangxi first introduced passion fruit in 1985, it has been guided by the government and planted spontaneously. It began to develop on a large scale around 2012. So far, the planting area and output of passion fruit have reached 432,000 mu and 361,000 tons respectively, both ranking first in the country, becoming Guangxi's advantageous and characteristic fruit industry.

[0003] However, while the scale of planting is rapidly expanding, there are still prominent problems in passion fruit planting, such as frequent occurrence of extreme high temperatures, which leads to limited flowering and fruiting in summer and low yields. From the perspective of the adaptability of conventional passion fruit varieties to the climate, the largest amount of flowers and fruits is in October-November; there is a certain amount of flowers and fruits in April-May and September, but there is greater uncertainty; there are few or even no flowers and fruits in the high temperature season from June to August; there are no flowers and fruits from December to March of the following year. Because the plants have a large number of branches but few flowers in summer, a large number of flowers in autumn and winter but the fruit matures too long, the fruit is small and of poor quality, resulting in a low yield of the main varieties of passion fruit in Guangxi. Therefore, the current increase in the production of commercial passion fruit requires breaking through the bottleneck of low flowers and fruits in the high temperature season. As the greenhouse effect causes global temperatures to rise and extreme weather increases, the entire planting industry will continue to face high temperature challenges.

[0004] In the traditional method, the heat resistance of passion fruit varieties is usually identified by observing the flowering and fruiting characteristics and the growth of plants in the field during the high temperature season, and the heat resistance of passion fruit varieties is identified based on the fact that the plants can bloom and set fruits normally during the high temperature period (maximum daily temperature>35°C). This identification method is relatively extensive, easily affected by factors such as the planting time and flowering period of the variety, lacks non-high temperature controls, and can only observe phenotypic agronomic traits. The plant's resistance to heat damage cannot be judged, so the application of traditional methods to identify the heat resistance of passion fruit is not comprehensive enough. Therefore, passion fruit production urgently needs to screen, identify and breed passion fruit varieties with heat resistance, but there is currently a lack of technical standards for the identification and evaluation of passion fruit heat resistance, which restricts the selection and breeding of new heat-resistant varieties and the promotion and application of superior heat-resistant varieties. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for identifying the heat resistance of passion fruit, so as to solve the problem of lack of a heat resistance identification and evaluation system during variety breeding and germplasm screening of passion fruit.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for identifying heat resistance of passion fruit comprises the following steps:

[0008] (1) Heat treatment: a high temperature treatment and a normal temperature control treatment were set for the passion fruit varieties to be identified. The high temperature treatment was a high temperature heat stress treatment in a plastic warming shed, and the normal temperature control treatment was a normal treatment under natural climatic conditions.

[0009] (2) Index detection: After the heat treatment, the relative conductivity, soluble protein content, soluble sugar content, superoxide dismutase activity, peroxidase activity and catalase activity of the plant leaves were measured;

[0010] And the pollen germination rate, the number of fruits per plant and the fruit setting rate of the plants were measured;

[0011] (3) Calculation of relative heat resistance index: Substitute the index measured in step (2) into the following formula to calculate the relative heat resistance index S value of the variety to be identified:

[0012]

[0013] F(Xi)=1-(Xi-Xmin) / (Xmax-Xmin);

[0014] F(Xi)=(Xi-Xmin) / (Xmax-Xmin);

[0015] S=∑F(Xi) / 9(i=1, 2, 3, 4, 5, 6, 7, 8, 9);

[0016] In the formula, H is the average value of the i-th index of the variety under high temperature heat stress treatment; is the average value of the ith index of all varieties to be identified under high temperature heat stress treatment; CK is the average value of the ith index of the variety under normal temperature control treatment; Xi is the heat resistance coefficient of the ith index of the variety; Xmin is the minimum value of the heat resistance coefficient of the ith index among all varieties to be identified; Xmax is the maximum value of the heat resistance coefficient of the ith index among all varieties to be identified;

[0017] (4) Ranking by heat resistance: Ranking the relative heat resistance index S values ​​of the varieties to be identified calculated in step (3), i.e., ranking the heat resistance of the corresponding varieties;

[0018] (5) Heat resistance classification: Based on the relative heat resistance index S value of known heat-resistant passion fruit varieties, the passion fruit heat resistance evaluation system is divided into four levels, as follows:

[0019]

[0020] (6) Heat resistance grade evaluation: The relative heat resistance index S value of the passion fruit variety to be identified calculated in step (3) is matched with the heat resistance grade in step (5) to evaluate the heat resistance grade of the variety to be identified.

[0021] Furthermore, in step (1), the high temperature treatment is: the maximum daily temperature is 38-45°C, the average daily temperature is 30-31°C, the daily temperature is ≥35°C for 4-6 hours, and the humidity in the greenhouse is >30%.

[0022] Furthermore, in step (1), the normal temperature control treatment is: the daily maximum temperature is less than 37°C, the daily average temperature is less than 28.5°C, and the daily temperature is ≥35°C for a duration of less than 2h.

[0023] Furthermore, in step (2), the relative conductivity of the leaves is determined by an immersion method; the soluble protein content of the leaves is determined by a Coomassie Brilliant Blue G-250 method; the soluble sugar content of the leaves is determined by an anthrone colorimetric method or a kit method; the superoxide dismutase activity of the leaves is determined by a nitrotetrazole blue chloride photoreduction method or a kit method; the peroxidase activity of the leaves is determined by a guaiacol method or a kit method; and the catalase activity of the leaves is determined by an ultraviolet spectrophotometric method or a kit method.

[0024] Furthermore, the leaf is the first fully expanded leaf of a new branch.

[0025] Furthermore, in step (2), the pollen germination rate is determined by collecting flower buds that bloom on the same day from the plants treated with high temperature and the plants treated with normal temperature as a control, removing the anthers from the flower buds at 24 to 26°C, and placing them in a ventilated and dry place for 1.5 to 2 hours. After the anthers are fully cracked, the pollen is swept down with a brush and mixed thoroughly, and then evenly sprinkled into the liquid culture medium and mixed thoroughly, and incubated in the dark at 24 to 26°C for 2.5 to 3 hours.

[0026] Furthermore, the formula of the liquid culture medium includes: 350-400 mg / mL Ca(NO3)2·4H2O, 80-100 g / L sucrose, 15-20 mg / L boric acid, and 130-150 g / L PEG-4000.

[0027] Furthermore, in step (6), H36 and MT were identified as highly heat-resistant varieties, H3, P5, P7 and Xia Ji were identified as moderately heat-resistant varieties, P1, Dong Hui Mi and Yuan Xiao were identified as intermediate varieties, and Huang You No. 2 and DN were identified as heat-sensitive varieties.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] 1. The method of the present invention evaluates the heat resistance of passion fruit varieties or germplasm resources, which is helpful for breeding and promoting heat-resistant varieties. The present invention is treated by high temperature and normal temperature control, combined with the determination of various physiological and growth indicators (such as relative conductivity, soluble protein content, soluble sugar content, antioxidant enzyme activity and pollen germination rate, single plant fruit number, fruit setting rate), can more accurately identify the heat resistance of passion fruit, calculate the relative heat resistance index S value from the above indicators, and sort and evaluate the heat resistance of the varieties according to the size of the relative heat resistance index S value. Thus, the heat resistance of passion fruit varieties or germplasm resources can be objectively and accurately evaluated, thereby contributing to the breeding of new heat-resistant varieties and the promotion and application of superior heat-resistant varieties.

[0030] 2. The present invention adopts a two-factor split-plot test design, with temperature treatment as the main plot factor to set high temperature treatment and normal temperature control treatment, and with the variety to be identified as the split-plot factor, to measure various physiological and growth indicators of passion fruit, and to calculate the relative heat resistance index S value of the variety to be identified from the above indicators using the membership function value method, wherein the formula is used Calculating the heat resistance coefficient Xi of the ith indicator of the variety can effectively avoid the deviation in the evaluation of the heat resistance of the variety when the indicator measurement value under high temperature treatment is higher than the control value but far lower than the average level of the group, and can more accurately identify the heat resistance of passion fruit.

[0031] 3. The heat resistance grade of passion fruit of the present invention is mainly based on the relative heat resistance index S value. The reasons are: (1) The larger the S value, the stronger the heat resistance. The heat resistance of the tested varieties can be ranked according to the S value. (2) Figure 3 It can be seen that the heat resistance classification of the varieties is consistent with the classification results of cluster analysis, which proves the reliability of the grade parameters and classification. (3) In the embodiment, a total of 12 varieties were tested. The S value and grade parameters obtained under the test conditions are also based on actual application results and can be used as a reference for the evaluation of the heat resistance grade of varieties under the same test conditions.

[0032] 4. The heat resistance grade of passion fruit divided with reference to the present invention is matched with the heat resistance grade of passion fruit according to the relative heat resistance index S value of known heat-resistant varieties. The obtained grading result corresponds to the heat resistance of the known varieties, which illustrates the reliability and accuracy of the identification method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the community distribution.

[0034] Figure 2 This is a schematic diagram of the community floor plan.

[0035] Figure 3 This is a pedigree diagram based on the systematic cluster analysis of the relative heat resistance index S value of the tested varieties. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below by way of examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0037] Example

[0038] In 2022, a scientific research base will be built at the Guangxi Academy of Agricultural Sciences in the ASEAN Economic Development Zone in Nanning, Guangxi to conduct an initial screening of the heat resistance of 50 passion fruit varieties (germplasm) resources planted in the open field. The screening criteria are: observing the heat damage and flowering and fruiting characteristics of passion fruit plants under high temperature conditions above 35°C. Due to the large number of heat-resistant plants identified in the planting observation of passion fruit hybrid offspring, the workload of later identification can be reduced by conducting an initial screening of heat resistance. The manifestation of weak heat resistance is weak plant growth or even wilting, and the inability to flower and set fruit normally; the manifestation of medium heat resistance is medium plant growth, few flowers, and low fruit setting rate; the manifestation of strong heat resistance is vigorous plant growth, normal flowering and fruiting. Twelve varieties (germplasm) resources, including MT, Huangyou No. 2, H36 (known to be highly heat-resistant varieties), Donghui Mi, Tainong No. 1 (known to be moderately heat-resistant varieties), Xia Ji, P1, P5, P7, H3, Yuanxiao and DN, were initially screened and entered the second stage of heat resistance identification.

[0039] Among the known heat-resistant varieties mentioned above, Tainong No. 1 is the main cultivated variety of purple passion fruit, which is recognized as a moderately heat-resistant variety in this field and is used as a reference variety. H36 is evaluated for heat resistance with reference to "8.2 Heat Resistance" of "8 Stress Resistance Traits" of Hainan Provincial Local Standard "DB46 / T 530-2021 Passion Fruit (Passion Fruit) Germplasm Resource Description Specification" and the method is as follows:

[0040] “Observe mature plants that have grown for more than 6 months and record their heat tolerance under natural conditions or high temperature stress environments, divided into:

[0041] - Weak (30% of the plants wilt at high temperatures above 35 degrees Celsius);

[0042] - Medium (under high temperature conditions above 35 degrees Celsius, leaves are burned and plants experience severe flower and fruit drop);

[0043] - Strong (under high temperature conditions above 35 degrees Celsius, the plants bloom and bear fruit normally)."

[0044] H36 meets the requirements of strong heat-resistant planting performance, and the inventors observed that the plants grew vigorously during the high temperature period for two consecutive years, and their flowering and fruiting characteristics were significantly better than other varieties. Therefore, it was classified as a highly heat-resistant variety in the initial heat resistance screening.

[0045] In 2023, 12 passion fruit varieties (germplasm) were selected to carry out the second phase of heat resistance identification test. A two-factor split-plot design was adopted, with temperature treatment as the main plot factor, including high temperature treatment and normal temperature control treatment, and variety as the split-plot factor. Planting was carried out on March 30, with 3 varieties planted in each plot, 2 to 3 plants of each variety, 1.8m in row spacing and 1.4m in plant spacing. High temperature treatment and normal temperature control were set up, repeated 3 times, and a total of 14 plants of each variety were planted. High temperature treatment and normal temperature control were set up. High temperature treatment method: Use steel pipes to make a high-temperature treatment growth shed frame with a length × width × height of 12.0m × 2.0m × 4.0m, fixed to the ditch surface, and at the beginning of the high temperature treatment, the temperature is raised by covering the four sides of the growth shed frame with plastic film (light transmittance of more than 95%). Leave a 20cm high gap evenly on the upper part of both sides of the greenhouse for gas exchange. After the high temperature treatment, the film will be lifted, and the later management and field microclimate will be consistent with the control. See the schematic diagram of the plot distribution. Figure 1 A two-factor split-plot design was used, with the main plot factor A1 being heat treatment and A2 being normal temperature control treatment; split-plot factors B1-B4 being varieties. Figure 2 A1 is the heat treatment of the plastic warming shed, and A2 is the normal temperature control treatment; the plastic warming shed is 2m wide and 12m long, the interval between adjacent warming sheds is 1.5m, the planting row width is 1.0m, and the planting is trellis-style.

[0046] Before heat treatment, the heating shed was fully opened and placed in the same natural environment as the control. When the secondary vines of the plants were pulled out, the high temperature heat stress treatment was started. The top and side films of the heating shed were lowered, and the temperature in the shed was monitored in real time until the high temperature setting value was reached, that is, the maximum temperature of the heat stress treatment day was 38-45℃, the daily average temperature was 30-31℃, the daily temperature was ≥35℃ for 4-6h, and the humidity in the shed was >30%; the maximum temperature of the natural control day was <37℃, the daily average temperature was <28.5℃, and the daily temperature was ≥35℃ for <2h. The heat treatment was terminated after 40 days.

[0047] Determination of relative conductivity of leaves: Sampling was performed after the heat stress treatment. The sampling site was the first fully expanded leaf of the new branch. The relative conductivity of the leaves was determined by the immersion method. Sampling was performed avoiding the spraying time. Each treatment was sampled and measured three times. After the leaves were collected and brought back to the laboratory in an ice box, the leaves were cut into pieces avoiding the leaf edges and veins. About 0.2g of the sample was immediately weighed and placed in a 20mL graduated test tube. 20mL of deionized water was added with a pipette. The leaves were pressed into the deionized water with a filter paper strip to submerge them. The leaves were sealed with plastic film and soaked at room temperature of about 26℃ for 16h. The filter paper was taken out the next day, and the liquid in the test tube was mixed several times. Part of the liquid was poured into a clean 10mL centrifuge tube to determine the initial conductivity. After the measurement, it was poured back into the original test tube and placed in a boiling water bath at 100℃ for 30min. After cooling to room temperature, the complete conductivity was determined. Relative conductivity = initial conductivity / complete conductivity*100%. The results are as follows:

[0048] Table 1 Relative conductivity (%) and membership function value F(Xi)

[0049]

[0050]

[0051] Note: H is the measured value under heat stress treatment, CK is the measured value of control, the same below.

[0052] Determination of soluble protein content: Each treatment was sampled and measured three times. The sampling site was the first fully expanded leaf of the new branch. The soluble protein content of the leaves was determined by the Coomassie Brilliant Blue G-250 method. 0.1g of fresh leaves were quickly weighed, put into a mortar, and 1mL of distilled water was added to grind to a homogenate. All were transferred to a centrifuge tube, centrifuged at 4000r / min for 20min, and the supernatant was taken for testing. 20μL of the extract and 5mL of G-250 reagent were added to the centrifuge tube, inverted to mix, and allowed to stand for 10min. A 1cm optical path cuvette was used to adjust the zero value with a blank tube at a wavelength of 595nm, and the absorbance value of each tube was determined. Preparation of standard curve: Take 0, 0.2, 0.4, 0.6, 0.8, 1.0mL of standard protein solution (100μg / mL), add distilled water to a total of 1.0mL, add 5mL of G-250 reagent to each tube, cover and invert to mix evenly, place for 10min, use a 1cm light path cuvette to zero at 595nm with a zero tube, and measure the absorbance of each tube, with protein content (μg) as the x-axis and absorbance value A of each tube as the y-axis. 595 As the y-axis, draw the standard curve and calculate the regression equation. Soluble protein content (mg / g fresh weight) = (x × V 提取 ) / (W×V 样 ). Where x is the soluble protein content read from the standard curve, V 提取 is the total volume of sample extract, V 样 is the volume of sample extract absorbed during the measurement, and W is the fresh weight of the sample. The results are as follows:

[0053] Table 2 Absorbance values ​​of each tube of the standard curve of soluble protein content

[0054] Serial number Protein content / μg <![CDATA[A 595 ]]> 1 0 0 2 20 0.158 3 40 0.285 4 60 0.398 5 80 0.498 6 100 0.636

[0055] Standard curve: y = 0.0062x + 0.0211 (R 2 =0.9953)

[0056] Table 3 Soluble protein content (mg / g fresh weight) and membership function value F(Xi)

[0057]

[0058]

[0059] Determination of soluble sugar content (kit method): Each treatment was sampled and measured three times. The sampling site was the first fully expanded leaf of the new branch. The plant soluble sugar content kit (catalog number JC0401-M, Nanjing JiCe Biotechnology Co., Ltd.) was used for determination. Weigh 0.05g of fresh leaf sample, add 1mL of distilled water and grind into a homogenate, pour into a covered centrifuge tube, and bathe at 95℃ for 10min (cover tightly to prevent water loss). After cooling, centrifuge at 8000g, 25℃ for 10min, and take the supernatant for testing (pay attention to dilution). Preheat the enzyme reader for more than 30min, adjust the wavelength to 620nm, and zero with distilled water. Adjust the water bath to 95℃. Add reagents as shown in the following table:

[0060] Table 4 Reagent addition

[0061] Reagent (μL) Blank tube Determination tube Standard tube sample 40 Standard solution 40 Distilled water 80 40 40 Working fluid 20 20 20 Concentrated sulfuric acid 200 200 200

[0062] After mixing, place in a 95℃ water bath for 10 min (cover tightly to prevent water loss), cool naturally to room temperature, transfer 200uL to an ELISA plate, record the absorbance value A at a wavelength of 620nm, and calculate ΔA 标准 =A 标准管 -A 空白管 , ΔA=A 测定管 -A 空白管 . (The blank tube only needs to be done 1-2 times).

[0063] Drawing of standard curve: With different concentrations of glucose standard solution as x-axis and ΔA standard as y-axis, draw the standard curve y=kx+b. The absorbance values ​​of each tube of the standard curve are as follows:

[0064] Table 5 Absorbance values ​​of each tube of standard curve

[0065]

[0066]

[0067] Standard curve: y = 3.6265x + 0.0176, R 2 =0.9986

[0068] Substitute the ΔA measurement into the equation to obtain the x value (mg / mL). Soluble sugar content (mg / g fresh weight) = x × V 提取 ÷W×dilution factor=x÷W×dilution factor. x is the soluble sugar content read on the standard curve; V 提取 is the total volume of sample extract, 1 mL; W is the fresh weight of the sample, and the dilution factor is 20 times. The results are as follows:

[0069] Table 6 Soluble sugar content (mg / g fresh weight) and membership function value F(Xi)

[0070] Variety (germplasm) H CK Xi Xmax Xmin F(Xi) MT 27.50 27.99 0.99 1.33 0.79 0.37 Huangyou No.2 24.11 26.77 0.89 1.33 0.79 0.18 H36 24.04 27.90 0.87 1.33 0.79 0.14 Winter Honey 24.89 22.89 0.99 1.33 0.79 0.38 Tainong No.1 29.81 18.93 1.33 1.33 0.79 1.00 Xia Ji 26.78 24.12 1.04 1.33 0.79 0.47 P1 32.45 35.35 1.05 1.33 0.79 0.48 P5 32.09 30.85 1.10 1.33 0.79 0.58 P7 34.95 31.78 1.18 1.33 0.79 0.73 H3 25.63 28.49 0.91 1.33 0.79 0.23 Small round 29.02 26.85 1.06 1.33 0.79 0.51 DN 20.86 25.28 0.79 1.33 0.79 0.00

[0071] Determination of superoxide dismutase activity: Each treatment was sampled and measured three times. The sampling site was the first fully expanded leaf of the new branch. The superoxide dismutase kit (Cat. No. JC0101-M, Nanjing JiCe Biotechnology Co., Ltd.) was used for determination. Weigh about 0.1g of fresh leaf sample, add 1mL of extract, and homogenize; centrifuge at 8000g, 4℃ for 10min, take the supernatant, and place on ice for testing. Preheat the microplate reader for more than 30min and adjust the wavelength to 560nm. Dilute reagent 2 twice with distilled water, and use as much as needed (reagent 2 and distilled water are diluted 1:1). Dissolve a bottle of reagent 4 with 5mL of distilled water (use it within one week after dissolution), and then dilute it 4 times with distilled water, and use as much as needed (reagent 4 and distilled water are diluted 1:3). Before determination, place reagents 1, 3, and 4 in a 25℃ water bath for more than 5min. Add the following reagents in a 96-well plate in sequence for sample determination:

[0072] Table 7 Reagent sample determination

[0073]

[0074]

[0075] Mix thoroughly, let stand at room temperature for 30 min, and measure the absorbance A of each tube at 560 nm.

[0076] Calculation of results: ① Calculation of inhibition percentage: Inhibition percentage = (A 对照管 -A 测定管 )÷A 对照管 ×100%. ②SOD enzyme activity unit: When the inhibition percentage in the above xanthine oxidase coupling reaction system is 50%, the SOD enzyme activity in the reaction system is defined as one enzyme activity unit (U / mL). ③SOD enzyme activity calculation: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1-inhibition percentage) × V 反总 ]÷(W×V 样 ÷V 样总 )=11.11×inhibition percentage÷(1-inhibition percentage)÷W. V 反总 : Total volume of reaction system, 0.2mL; V 样 : Sample volume added to the reaction system, 0.018mL; V 样总 : Volume of extract added, 1mL; W: Sample mass, g. The results are as follows:

[0077] Table 8 Superoxide dismutase activity (U / g fresh weight) and membership function value F(Xi)

[0078] Variety (germplasm) H CK Xi Xmax Xmin F(Xi) MT 353.23 403.38 0.93 1.27 0.83 0.22 Huangyou No.2 423.46 476.36 1.03 1.27 0.83 0.46 H36 466.61 373.45 1.27 1.27 0.83 1.00 Winter Honey 277.69 183.09 1.14 1.27 0.83 0.71 Tainong No.1 319.84 413.69 0.83 1.27 0.83 0.00 Xia Ji 412.55 444.59 1.04 1.27 0.83 0.47 P1 383.16 416.55 0.99 1.27 0.83 0.36 P5 333.91 355.51 0.93 1.27 0.83 0.23 P7 296.77 297.84 0.91 1.27 0.83 0.18 H3 388.52 440.60 0.98 1.27 0.83 0.34 Small round 309.22 322.42 0.91 1.27 0.83 0.18 DN 366.37 307.08 1.10 1.27 0.83 0.62

[0079] Peroxidase activity determination: Each treatment was sampled and measured three times, and the sampling site was the first fully expanded leaf of the new branch. The peroxidase kit (Cat. No. JC0102-S, Nanjing JiCe Biotechnology Co., Ltd.) was used for determination. Weigh 0.1g of fresh leaf sample, add 1mL of extract, and homogenize; centrifuge at 8000g, 4℃ for 10min, take the supernatant, and place it on ice for testing. Preheat the spectrophotometer for more than 30min, adjust the wavelength to 470nm, and adjust the zero with distilled water. Preparation of working solution: Mix reagent 1, reagent 2, and reagent 3 in a ratio of 2.6mL:1.5μL:1μL before use; preheat at 25℃ for more than 10min; prepare and use immediately. Add 50μL of sample and 950μL of working solution to a 1mL glass cuvette, mix, and record the absorbance value A1 at 470nm for 1min and the absorbance value A2 after 2min. Calculate ΔA=A2-A1.

[0080] Result calculation: POD activity unit definition: A per gram of tissue in each mL of reaction system per minute 470 A change of 0.01 is one unit of enzyme activity. POD (U / g fresh weight) = ΔA × V 反总 ÷(W×V 样 ÷V 样总 )÷0.01÷T=2000×ΔA÷W. V 反总 : Total volume of reaction system, 1mL; V 样 : Added sample volume, 0.05mL; V 样总 : Volume of extract added, 1 mL; T: reaction time, 1 min; W: fresh weight of sample. The results are as follows:

[0081] Table 9 Peroxidase activity (U / g fresh weight) and membership function value F(Xi)

[0082] Variety (germplasm) H CK Xi Xmax Xmin F(Xi) MT 2233.74 2044.82 1.44 1.66 0.25 0.84 Huangyou No.2 2194.44 3138.03 1.23 1.66 0.25 0.69 H36 2652.47 2219.13 1.66 1.66 0.25 1.00 Winter Honey 393.63 1534.88 0.29 1.66 0.25 0.03 Tainong No.1 225.99 386.90 0.38 1.66 0.25 0.10 Xia Ji 669.86 365.61 1.18 1.66 0.25 0.66 P1 1182.69 1894.93 0.79 1.66 0.25 0.38 P5 1728.97 1432.69 1.30 1.66 0.25 0.74 P7 1006.46 1067.42 0.88 1.66 0.25 0.44 H3 1222.22 2584.70 0.73 1.66 0.25 0.34 Small round 1143.64 1735.85 0.79 1.66 0.25 0.38 DN 304.52 1215.72 0.25 1.66 0.25 0.00

[0083] Catalase activity determination: Each treatment was sampled and measured three times. The sampling site was the first fully expanded leaf of the new branch. The catalase kit (Cat. No. JC0103-S, Nanjing JiCe Biotechnology Co., Ltd.) was used for determination. About 0.1g of fresh leaf sample was weighed, 1mL of extract was added, and homogenized; 8000g, centrifuged at 4℃ for 10min, the supernatant was taken, and placed on ice for testing. The microplate reader was preheated for more than 30min, the wavelength was adjusted to 240nm, and distilled water was adjusted to zero. Before the determination, the CAT detection working solution was bathed at 25℃ for more than 10min. Prepare a 96-well UV plate. Add 10μL of sample and 190μL of working solution to the microplate plate (UV plate), mix well, and immediately measure the initial absorbance value A1 at 240nm and the absorbance value A2 after 1min at room temperature, and calculate ΔA=A1-A2.

[0084] Calculation of CAT activity: Definition of CAT activity unit: One unit of enzyme activity is defined as the degradation of 1 μmol H2O2 per minute per milliliter of sample extract. CAT (U / mL) = [ΔA × V 反总 ÷(ε×d)×10 6 ]÷V 样 ÷T.V 反总 : Total volume of reaction system, 1.035×10 -3 L; ε: H2O2 molar extinction coefficient, 43.6L / mol / cm; d: cuvette light path, 1cm; V 样 : added sample volume, 0.035mL; T: reaction time, 1min; 10 6 : Unit conversion factor, 1mol = 10 6 μmol. The results are as follows:

[0085] Table 10 Catalase activity (U / g fresh weight) and membership function value F(Xi)

[0086]

[0087]

[0088] Pollen germination rate determination: After 40 days of heat stress treatment, collect the flower buds (the flower buds have turned white, and the petals have not yet unfolded) of the heat stress treatment and control plants that bloomed on the same day. Collect 8 flowers from each plot for each treatment, and collect 24 flowers from 3 plots. Bring the flower buds back to the laboratory, remove the anthers and place them on weighing paper in a 25°C environment, and place them in a ventilated and dry place for 2 hours. When the anthers are fully cracked, use a brush to sweep the pollen and mix them thoroughly for use. Take a 5cm diameter culture dish, drop 4.0mL of liquid culture medium into the culture dish, sprinkle the pollen sample evenly into the culture dish and mix it thoroughly, culture it in the liquid culture medium at 25°C in the dark for 3 hours, and count the number of pollen germination in 5 fields of view under an optical microscope. The total number of pollen in each field of view is ≥60 grains. Repeat the measurement 3 times for each sample, and calculate the pollen germination rate (%). The liquid culture medium formula is: 400mg / mL Ca(NO3)2·4H2O, 100g / L sucrose, 20mg / L boric acid (H3BO3), 150g / L PEG-4000. The results are as follows:

[0089] Table 11 Pollen germination rate (%) and membership function value F(Xi)

[0090]

[0091]

[0092] Determination of the number of fruits per plant: The number of fruits per plant in heat stress treatment and control plants was counted from the beginning to the end of heat stress. All plants in each plot were counted and repeated 3 times. The results are as follows:

[0093] Table 12 Number of fruits per plant (pieces / plant) and membership function value F(Xi)

[0094] Variety (germplasm) H CK Xi Xmax Xmin F(xi) MT 12.00 25.70 1.95 1.95 0.00 1.00 Huangyou No.2 0.00 0.89 0.00 1.95 0.00 0.00 H36 7.57 13.86 1.35 1.95 0.00 0.70 Winter Honey 0.00 0.00 0.00 1.95 0.00 0.00 Tainong No.1 2.29 4.43 0.58 1.95 0.00 0.30 Xia Ji 5.14 14.14 0.92 1.95 0.00 0.47 P1 2.43 2.86 0.77 1.95 0.00 0.40 P5 2.86 3.14 0.86 1.95 0.00 0.44 P7 4.14 3.00 1.28 1.95 0.00 0.66 H3 4.43 5.29 1.05 1.95 0.00 0.54 Small round 0.00 1.43 0.00 1.95 0.00 0.00 DN 1.14 2.00 0.45 1.95 0.00 0.23

[0095] Determination of fruit setting rate: Investigate the fruit setting rate of heat stress treatment and control plants during the flowering period. Select the flowers that bloomed on the same day to hang the date, and hang the flowers for 3 to 5 consecutive days. The number of flowers hung in each plot is ≥50, and repeated 3 times. The number of flowers hung in each treatment is ≥150; investigate the fruit setting situation 20 days after hanging, count the number of fruits, and calculate the fruit setting rate (%) = number of fruits / number of hanging flowers. The results are as follows:

[0096] Table 13 Fruit setting rate (%) and membership function value F(Xi)

[0097]

[0098]

[0099] Calculation of relative heat resistance index of tested passion fruit varieties (germplasm): Calculate the relative heat resistance index of each measured index according to the following formula:

[0100]

[0101] F(Xi)=1-(Xi-Xmin) / (Xmax-Xmin);

[0102] F(Xi)=(Xi-Xmin) / (Xmax-Xmin);

[0103] S=∑F(Xi) / 9(i=1, 2, 3, 4, 5, 6, 7, 8, 9);

[0104] In the formula, H is the average value of the i-th index of the variety under high temperature heat stress treatment; is the average value of the ith index of all varieties to be identified under high temperature heat stress treatment; CK is the average value of the ith index of the variety under normal temperature control treatment; Xi is the heat resistance coefficient of the ith index of the variety; Xmin is the minimum value of the heat resistance coefficient of the ith index among all varieties; Xmax is the maximum value of the heat resistance coefficient of the ith index among all varieties. The results are as follows:

[0105] Table 14 Relative heat resistance index S

[0106]

[0107] The varieties are ranked according to their relative heat resistance index S value. The larger the S value, the stronger the heat resistance of the variety. The heat resistance ranking of the 12 participating passion fruit varieties is shown in Table 14.

[0108] Based on the above heat resistance index S value, the passion fruit heat resistance evaluation system is divided into four levels, as follows:

[0109]

[0110] Referring to the above-mentioned passion fruit heat tolerance evaluation and grading, it can be seen from the results of the relative heat tolerance index S value that among the 12 passion fruit varieties (germplasm) resources participating in the test in 2023, there are 2 varieties (germplasm) resources identified as highly heat-resistant: H36 and MT, 5 moderately heat-resistant germplasm resources: H3, Tainong No. 1, P5, P7, Xia Ji, 3 intermediate varieties (germplasm) resources: P1, Donghuimi and Yuanxiao, and 2 heat-sensitive varieties (germplasm) resources: Huangyou No. 2 and DN.

[0111] The above heat resistance grades are mainly divided based on the relative heat resistance index S value. The reasons are: (1) The larger the S value, the stronger the heat resistance. The heat resistance of the tested varieties can be ranked according to the S value. (2) Figure 3It can be seen that the heat resistance classification of the varieties is consistent with the classification results of cluster analysis, which proves the reliability of the grade parameters and classification. (3) In the embodiment, a total of 12 varieties were tested. The S value and grade parameters obtained under the test conditions are also based on actual application results and can be used as a reference for the evaluation of the heat resistance grade of varieties under the same test conditions.

[0112] According to the previously known fact that H36 and Tainong No. 1 are highly heat-resistant and moderately heat-resistant varieties, respectively, and the relative heat resistance index S values ​​of H36 and Tainong No. 1 calculated by the identification method of the present invention are 0.69 and 0.53, respectively. By matching these two relative heat resistance index S values ​​with the heat resistance grade of passion fruit, it can be known that H36 and Tainong No. 1 are highly heat-resistant and moderately heat-resistant varieties, which corresponds to the previously known heat resistance of H36 and Tainong No. 1, illustrating the reliability and accuracy of the identification method of the present invention.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying the heat resistance of passion fruit, characterized in that: The following steps are involved: (1) Heat treatment: a high temperature treatment and a normal temperature control treatment were set for the passion fruit varieties to be identified. The high temperature treatment was a high temperature heat stress treatment in a plastic warming shed, and the normal temperature control treatment was a normal treatment under natural climatic conditions. (2) Index detection: After the heat treatment, the relative conductivity, soluble protein content, soluble sugar content, superoxide dismutase activity, peroxidase activity and catalase activity of the plant leaves were measured; And the pollen germination rate, the number of fruits per plant and the fruit setting rate of the plants were measured; (3) Calculation of relative heat resistance index: Substitute the index measured in step (2) into the following formula to calculate the relative heat resistance index S value of the variety to be identified: F(Xi)=1-(Xi-Xmin) / (Xmax-Xmin); F(Xi)=(Xi-Xmin) / (Xmax-Xmin); S=∑F(Xi) / 9(i=1, 2, 3, 4, 5, 6, 7, 8, 9); In the formula, H is the average value of the i-th index of the variety under high temperature heat stress treatment; is the average value of the ith index of all varieties to be identified under high temperature heat stress treatment; CK is the average value of the i-th index of the variety under the normal temperature control treatment; Xi is the heat resistance coefficient of the i-th index of the variety; Xmin is the minimum value of the heat resistance coefficient of the i-th index among all the varieties to be identified; Xmax is the maximum value of the heat resistance coefficient of the i-th index among all the varieties to be identified; (4) Ranking by heat resistance: Ranking the relative heat resistance index S values ​​of the varieties to be identified calculated in step (3), i.e., ranking the heat resistance of the corresponding varieties; (5) Heat resistance classification: Based on the relative heat resistance index S value of known heat-resistant passion fruit varieties, the passion fruit heat resistance evaluation system is divided into four levels, as follows: (6) Heat resistance grade evaluation: The relative heat resistance index S value of the passion fruit variety to be identified calculated in step (3) is matched with the heat resistance grade in step (5) to evaluate the heat resistance grade of the variety to be identified.

2. The method for identifying heat resistance of passion fruit according to claim 1, wherein In step (1), the high temperature treatment is as follows: the maximum daily temperature is 38-45°C, the average daily temperature is 30-31°C, the daily temperature is ≥35°C for 4-6 hours, and the humidity in the greenhouse is >30%.

3. The method for identifying heat resistance of passion fruit according to claim 1, wherein In step (1), the normal temperature control treatment is: the daily maximum temperature is less than 37°C, the daily average temperature is less than 28.5°C, and the daily temperature is ≥35°C for a duration of less than 2h.

4. The method for identifying heat resistance of passion fruit according to claim 1, wherein In step (2), the relative conductivity of the leaves is determined by an immersion method; the soluble protein content of the leaves is determined by a Coomassie Brilliant Blue G-250 method; the soluble sugar content of the leaves is determined by an anthrone colorimetric method or a kit method; the superoxide dismutase activity of the leaves is determined by a nitrotetrazole blue chloride photoreduction method or a kit method; the peroxidase activity of the leaves is determined by a guaiacol method or a kit method; and the catalase activity of the leaves is determined by an ultraviolet spectrophotometric method or a kit method.

5. The method for identifying heat resistance of passion fruit according to claim 4, wherein: The leaf is the first fully expanded leaf of a new branch.

6. The method for identifying heat resistance of passion fruit according to claim 1, wherein: In step (2), the pollen germination rate is determined by collecting flower buds that bloom on the same day from the plants treated with high temperature and the plants treated with normal temperature, removing the anthers from the flower buds at 24 to 26°C, and placing them in a ventilated and dry place for 1.5 to 2 hours. After the anthers are fully cracked, the pollen is swept down with a brush and mixed thoroughly, and then evenly sprinkled into the liquid culture medium and mixed thoroughly, and incubated in the dark at 24 to 26°C for 2.5 to 3 hours.

7. The method for identifying heat resistance of passion fruit according to claim 6, wherein: The formula of the liquid culture medium includes: 350-400 mg / mL Ca(NO3)2·4H2O, 80-100 g / L sucrose, 15-20 mg / L boric acid, and 130-150 g / L PEG-4000.

8. The method for identifying heat resistance of passion fruit according to claim 1, wherein: In step (6), H36 and MT were identified as highly heat-resistant varieties, H3, P5, P7 and Xia Ji were identified as moderately heat-resistant varieties, P1, Dong Hui Mi and Yuan Xiao were identified as intermediate varieties, and Huang You No. 2 and DN were identified as heat-sensitive varieties.

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