Method for identifying drought-enduring germplasm material of Chinese cabbage

Abstract

The invention relates to the field of crop germplasm resource identification and breeding, in particular to an identification method of a drought-enduring germplasm material of Chinese cabbage. The method comprises the following steps: firstly, preparing to-be-identified Chinese cabbage germplasm materials, performing germination period identification, and measuring the relative root length, the relative seedling height, the germination drought resistance index and the relative vitality index of seeds of each Chinese cabbage germplasm material; then seedling stage identification is carried out, and the relative plant fresh weight, the relative water content decreasing amplitude, the specific leaf weight decreasing amplitude, the damage rate, the in-vitro leaf water loss rate decreasing amplitude, the malondialdehyde content increasing amplitude and the proline content increasing amplitude of plants in the five-leaf stage of each Chinese cabbage germplasm material are measured; the membership function value and the average membership function value of each index are calculated in the germination stage and the seedling stage, and the drought tolerance is divided into five levels according to the average membership function value; and finally, comprehensively identifying. The method solves the problems that in the prior art, drought-tolerant planting is carried out only by optimizing an irrigation technology in the drought tolerance research of Chinese cabbages, and the identification and breeding research on the drought tolerance of varieties are few.

Description

Technical Field

[0001] The present invention relates to the field of identification and breeding of crop germplasm resources, and specifically to a method for identifying drought-tolerant germplasm materials of Chinese cabbage. Background Technique

[0002] Chinese cabbage (Brassica rapa subsp. pekinensis) belongs to the genus Brassica of the Brassicaceae family and is the second largest crop in terms of the planting area of open-field vegetables in China. The root system of Chinese cabbage is underdeveloped, and the environment needs to be kept moist throughout the growth period. In case of drought stress, the contents of polyphenols, flavonoids, total antioxidant enzyme activity, etc. will be significantly affected, resulting in poor growth and development, increased fiber, etc.

[0003] Traditional Chinese cabbage production requires a large amount of water, and the field management cost is relatively high. At present, the research on the drought tolerance of Chinese cabbage only conducts drought-tolerant planting by optimizing irrigation techniques, while there is little research on the identification and breeding of variety drought tolerance.

[0004] Based on this, it is necessary to invent a method for identifying drought-tolerant germplasm materials of Chinese cabbage. Summary of the Invention

[0005] In order to solve the problem that the current research on the drought tolerance of Chinese cabbage only conducts drought-tolerant planting by optimizing irrigation techniques, while there is little research on the identification and breeding of variety drought tolerance, the present invention provides a method for identifying drought-tolerant germplasm materials of Chinese cabbage.

[0006] The present invention is implemented by adopting the following technical scheme:

[0007] A method for identifying drought-tolerant germplasm materials of Chinese cabbage includes the following steps:

[0008] S1: Prepare the Chinese cabbage germplasm materials to be identified: Select the seeds of different Chinese cabbage germplasm materials and the 5-leaf stage plants corresponding to these Chinese cabbage germplasm materials;

[0009] S2: Identification at the germination stage: Perform drought stress treatment on the seeds of each Chinese cabbage germplasm material and set a control group, measure the four indexes of relative root length, relative seedling height, germination drought resistance index and relative vigor index of the seeds of each Chinese cabbage germplasm material, calculate the membership function values and average membership function values of each index, and divide the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value;

[0010] S3: Seedling stage identification: For the 5-leaf stage plants of each Chinese cabbage germplasm material, conduct drought stress treatment at the seedling stage and set up a control group. Measure the following seven indicators of the 5-leaf stage plants of each Chinese cabbage germplasm material: relative plant fresh weight, relative water content reduction, specific leaf weight reduction, injury rate, in vitro leaf water loss rate reduction, malondialdehyde content increase, and proline content increase. Calculate the membership function values and average membership function values of each indicator, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function values;

[0011] S4: Comprehensive identification: Screen the Chinese cabbage germplasm materials with the identification results of level 1 in both the germination stage and the seedling stage, and determine them as drought-tolerant germplasm materials.

[0012] Furthermore, in step S2, the drought stress treatment at the germination stage includes: Place the seeds of each Chinese cabbage germplasm material in a petri dish containing 10 mL of polyethylene glycol solution with a solution concentration of 15%, cover the petri dish lid, and place it in an environment with a temperature of 25°C, 12 h of light and 12 h of darkness for continuous cultivation for 8 days. Observe and measure the germination of these seeds every day, and consider the emergence of the seed coat as germination; The molecular weight of the polyethylene glycol is 6000 Da; The solution concentration is the mass-volume ratio.

[0013] Furthermore, in step S2, the calculation formula for the relative root length is:

[0014] Relative root length = root length after drought stress treatment at the germination stage / root length of the control group × 100%;

[0015] The calculation formula for the relative seedling height is:

[0016] Relative seedling height = seedling height after drought stress treatment at the germination stage / seedling height of the control group × 100%.

[0017] Furthermore, in step S2, the calculation formula for the germination drought resistance index is:

[0018] Germination drought resistance index = seed germination index after drought stress treatment at the germination stage / seed germination index of the control group;

[0019] Among them, the calculation formula for the seed germination index is:

[0020] Seed germination index = 1.00×Rd2 + 0.75×Rd4 + 0.50×Rd6 + 0.25×Rd8;

[0021] In the formula:

[0022] Rd2, Rd4, Rd6, and Rd8 are the germination rates on the 2nd, 4th, 6th, and 8th days respectively;

[0023] 1.00, 0.75, 0.50, and 0.25 are the drought resistance coefficients assigned to the corresponding germination days respectively;

[0024] The calculation formula for the relative vigor index is as follows:

[0025] Relative vigor index = vigor index after drought stress treatment during germination / vigor index of the control group × 100%;

[0026] Among them, the calculation formula for the vigor index is as follows:

[0027] Vigor index = radicle length × germination index;

[0028] Among them, the calculation formula for the germination index is as follows:

[0029]

[0030] In the formula:

[0031] Gi is the number of germinations on the i-th day;

[0032] Di is the corresponding germination day, that is, Di = i;

[0033] n is the number of experimental days, that is, n = 8.

[0034] Furthermore, in step S3, the seedling stage drought stress treatment includes: putting the 5-leaf stage plants of each Chinese cabbage germplasm material into a beaker with roots and substrate, standing for 12 h, pouring 80 mL of polyethylene glycol solution with a solution concentration of 10% into the beaker, and placing it at room temperature for 24 h; the molecular weight of the polyethylene glycol is 6000 Da; the solution concentration is the mass-to-volume ratio.

[0035] Furthermore, in step S3, the calculation formula for the relative plant fresh weight is as follows:

[0036] Relative plant fresh weight = plant fresh weight after drought stress treatment during the seedling stage / plant fresh weight of the control group;

[0037] The calculation formula for the relative water content reduction is as follows:

[0038] Relative water content reduction = (relative water content after drought stress treatment during the seedling stage - relative water content of the control group) / relative water content of the control group × 100%;

[0039] Among them, the calculation formula for the relative water content is as follows:

[0040] Relative water content = (leaf fresh weight - leaf dry weight) / (leaf saturated water weight - leaf dry weight).

[0041] Furthermore, in step S3, the calculation formula for the specific leaf weight reduction is as follows:

[0042] Specific leaf weight decrease rate = (Specific leaf weight after seedling stage drought stress treatment - Specific leaf weight of the control group) / Specific leaf weight of the control group × 100%;

[0043] Among them, the calculation formula for the specific leaf weight is:

[0044] Specific leaf weight = Leaf weight / Leaf area;

[0045] The calculation formula for the injury rate is:

[0046] Injury rate = (Conductivity value after seedling stage drought stress treatment - Conductivity value of the control group) / (Boiled conductivity value after seedling stage drought stress treatment - Conductivity value of the control group) × 100%.

[0047] Furthermore, in step S3, the calculation formula for the reduction rate of water loss rate of detached leaves is:

[0048] Reduction rate of water loss rate of detached leaves = (Water loss rate of detached leaves after seedling stage drought stress treatment - Water loss rate of detached leaves of the control group) / Water loss rate of detached leaves of the control group × 100%;

[0049] The calculation formula for the increase rate of malondialdehyde content is:

[0050] Increase rate of malondialdehyde content = (Malondialdehyde content after seedling stage drought stress treatment - Malondialdehyde content of the control group) / Malondialdehyde content of the control group × 100%;

[0051] The calculation formula for the increase rate of proline content is:

[0052] Increase rate of proline content = (Proline content after seedling stage drought stress treatment - Proline content of the control group) / Proline content of the control group × 100%.

[0053] Furthermore, the calculation method of the membership function value is:

[0054] For the indicators positively correlated with drought tolerance, namely relative root length, relative seedling height, germination drought resistance index, relative vigor index, relative plant fresh weight, the formula U(X i ) = (X i - X min ) / (X max - X min ) is used;

[0055] For the indicators negatively correlated with drought tolerance, namely relative water content decrease rate, specific leaf weight decrease rate, injury rate, reduction rate of water loss rate of detached leaves, increase rate of malondialdehyde content, increase rate of proline content, the formula U(X i ) = 1 - (X i - X min ) / (X max-X min )

[0056] Wherein:

[0057] U(X i ) is the membership function value of the i-th Chinese cabbage germplasm material to be identified for this index;

[0058] X i is the measured value of this index of the i-th Chinese cabbage germplasm material to be identified, and i corresponds to the number of different Chinese cabbage germplasm materials;

[0059] X max is the maximum value among the measured values of this index of all Chinese cabbage germplasm materials to be identified;

[0060] X min is the minimum value among the measured values of this index of all Chinese cabbage germplasm materials to be identified;

[0061] The calculation method of the average membership function value is as follows:

[0062]

[0063] Wherein:

[0064] is the average membership function value of n indexes of the i-th Chinese cabbage germplasm material to be identified;

[0065] n is the number of measured index items.

[0066] Furthermore, the grading standard of drought tolerance is as follows:

[0067] Grade 1 is drought-tolerant, that is

[0068] Grade 2 is relatively drought-tolerant, that is

[0069] Grade 3 is moderately drought-tolerant, that is

[0070] Grade 4 is drought-sensitive, that is

[0071] Grade 5 is extremely drought-sensitive, that is

[0072] Where is the average membership function value of n indexes of the i-th Chinese cabbage germplasm material to be identified.

[0073] The present invention provides a method for identifying drought-tolerant germplasm materials of Chinese cabbage. By simulating a drought environment with a polyethylene glycol solution at a concentration of 15% during the germination period of Chinese cabbage germplasm materials and measuring four indicators, and by simulating a drought environment with a polyethylene glycol solution at a concentration of 10% during the seedling stage and measuring seven indicators for dual screening, combined with membership function analysis and hierarchical evaluation, a comprehensive identification system for the drought tolerance of Chinese cabbage germplasm is established. This multi-stage and multi-parameter collaborative evaluation significantly improves the accuracy and reliability of the identification results. At the same time, by comprehensively selecting materials that reach drought tolerance level 1 during both the germination period and the seedling stage, it can be directly used in breeding practice, avoiding the time-consuming and laborious problems relying solely on field trials. In addition, traditional methods rely on optimizing irrigation techniques rather than variety improvement, while the present invention realizes the transformation from "passive drought resistance" to "active breeding of drought-tolerant varieties" by establishing a standardized identification system, providing an efficient tool for screening drought-tolerant germplasm resources and breeding varieties of Chinese cabbage. Detailed implementation mode

[0074] The present invention will be further described in detail below through specific embodiments. It should be noted that the present invention is not limited to the following embodiments.

[0075] A method for identifying drought-tolerant germplasm materials of Chinese cabbage includes the following steps:

[0076] S1: Prepare the Chinese cabbage germplasm materials to be identified: Select the seeds of 100 different Chinese cabbage germplasm materials and the 5-leaf stage plants corresponding to these Chinese cabbage germplasm materials, and number these 100 different Chinese cabbage germplasm materials as B1 to B100 respectively.

[0077] S2: Identification during the germination period: Carry out drought stress treatment on the seeds of each Chinese cabbage germplasm material and set a control group, measure the four indicators of relative root length, relative seedling height, germination drought resistance index, and relative vigor index of the seeds of each Chinese cabbage germplasm material, calculate the membership function values and average membership function values of each indicator, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value.

[0078] S201: First, polyethylene glycol (PEG-6000) with a molecular weight of 6000 Da was used to simulate drought stress. Specifically, a polyethylene glycol solution with a concentration of 15% was prepared, and the solution concentration was the mass-to-volume ratio (w / v). Then, for 100 Chinese cabbage germplasm materials, 10 seeds of uniform size, plump and healthy were selected for each material and placed in a petri dish with a diameter of 9 cm lined with double filter papers. 10 mL of the polyethylene glycol solution with a concentration of 15% was measured with a graduated cylinder and poured into each petri dish respectively. After covering the petri dish lid, it was continuously cultured for 8 days in an environment with a temperature of 25 °C and 12 h of light and 12 h of darkness each; a control group was set for each material. The difference between the control group and the drought stress group was that the polyethylene glycol solution in the petri dish was replaced with distilled water, and other test conditions were the same; the germination of the seeds of these 100 Chinese cabbage germplasm materials was observed and measured daily. The seeds were considered germinated when the radicle emerged. After 8 days of drought stress treatment, the indexes of the seeds of these 100 Chinese cabbage germplasm materials were measured.

[0079] S202: Measure the relative root length of the seeds of each Chinese cabbage germplasm material. The calculation formula for the relative root length is:

[0080] Relative root length = root length after drought stress treatment during germination period / root length of the control group × 100%;

[0081] In the formula: the units of the root length after drought stress treatment during germination period and the root length of the control group are both cm.

[0082] S203: Measure the relative shoot height of the seeds of each Chinese cabbage germplasm material. The calculation formula for the relative shoot height is:

[0083] Relative shoot height = shoot height after drought stress treatment during germination period / shoot height of the control group × 100%;

[0084] In the formula: the units of the shoot height after drought stress treatment during germination period and the shoot height of the control group are both cm.

[0085] S204: Measure the germination drought resistance index of the seeds of each Chinese cabbage germplasm material. The calculation formula for the germination drought resistance index is:

[0086] Germination drought resistance index = seed germination index after drought stress treatment during germination period / seed germination index of the control group;

[0087] Among them, the calculation formula for the seed germination index is:

[0088] Seed germination index = 1.00 × Rd2 + 0.75 × Rd4 + 0.50 × Rd6 + 0.25 × Rd8;

[0089] In the formula:

[0090] Rd2, Rd4, Rd6, and Rd8 are the germination rates on the 2nd, 4th, 6th, and 8th days respectively;

[0091] 1.00, 0.75, 0.50, and 0.25 are the drought resistance coefficients assigned to the corresponding germination days.

[0092] S205: Determine the relative vigor index of the seeds of each Chinese cabbage germplasm material. The calculation formula for the relative vigor index is:

[0093] Relative vigor index = vigor index after drought stress treatment during germination period / vigor index of the control group × 100%;

[0094] Among them, the calculation formula for the vigor index is:

[0095] Vigor index = radicle length × germination index;

[0096] In the formula: The unit of the radicle length is cm;

[0097] Among them, the calculation formula for the germination index is:

[0098]

[0099] In the formula:

[0100] Gi is the number of germinations on the i-th day, and the unit is individual;

[0101] Di is the corresponding germination day, that is, Di = i, and the unit is day;

[0102] n is the number of experimental days, n = 8, and the unit is day;

[0103] All relative indicators are the ratios of the drought stress treatment group to the control group.

[0104] S206: Calculate the membership function values and average membership function values of each index, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value.

[0105] The calculation method of the membership function value is:

[0106] For the indicators positively correlated with drought tolerance, namely relative root length, relative seedling height, germination drought resistance index, and relative vigor index, the formula U(X i ) = (X i - X min ) / (X max - X min ) is adopted;

[0107] In the formula:

[0108] U(X i) is the membership function value of this index in the i-th Chinese cabbage germplasm material to be identified;

[0109] X i is the measured value of this index of the i-th Chinese cabbage germplasm material to be identified, and i is B1, B2, B3... B100;

[0110] X max is the maximum value among the measured values of this index in the 100 Chinese cabbage germplasm materials to be identified;

[0111] X min is the minimum value among the measured values of this index in the 100 Chinese cabbage germplasm materials to be identified;

[0112] The calculation method of the average membership function value is as follows:

[0113]

[0114] In the formula:

[0115] is the average membership function value of 4 indexes of the i-th Chinese cabbage germplasm material to be identified;

[0116] n = 4;

[0117] The described X i and X max and X min have the same unit;

[0118] The classification standard of drought tolerance is as follows:

[0119] Level 1 is drought-tolerant, that is

[0120] Level 2 is relatively drought-tolerant, that is

[0121] Level 3 is moderately drought-tolerant, that is

[0122] Level 4 is drought-sensitive, that is

[0123] Level 5 is extremely drought-sensitive, that is

[0124] The results of the four indexes of relative root length, relative seedling height, germination drought resistance index and relative vigor index measured in steps S202 to S206 and the average membership function value of these four indexes are shown in Table 1, and the drought tolerance at the germination stage of these 100 Chinese cabbage germplasm materials is divided into five levels.

[0125] Level 1, Drought-tolerant materials: B19, B28, B33, B49, B59, B94, a total of 6, accounting for 6%;

[0126] Level 2, Relatively drought-tolerant materials: B20, B27, B37, B39, B41, B46, B51, B62, B73, B91, B96, B100, a total of 12, accounting for 12%;

[0127] Level 3, Moderately drought-tolerant materials: B2, B4, B6, B7, B8, B9, B10, B12, B13, B21, B23, B25, B26, B31, B34, B35, B36, B38, B40, B42, B44, B45, B47, B48, B50, B52, B53, B54, B56, B57, B58, B61, B66, B69, B70, B71, B74, B75, B77, B81, B82, B85, B88, B89, B90, B92, B95, a total of 47, accounting for 47%;

[0128] Level 4, Drought-sensitive materials: B1, B3, B5, B11, B14, B15, B16, B17, B18, B22, B24, B29, B30, B32, B43, B55, B60, B64, B65, B67, B68, B72, B76, B78, B79, B80, B83, B84, B86, B87, B93, B98, B99, a total of 33, accounting for 33%;

[0129] Level 5, Extremely drought-sensitive materials: B63, B97, a total of 2, accounting for 2%.

[0130] Table 1 Membership function values and comprehensive evaluation table of drought tolerance indexes of 100 Chinese cabbage germplasm materials at germination stage

[0131]

[0132]

[0133]

[0134] S3: Seedling stage identification: For the 5-leaf stage plants of each Chinese cabbage germplasm material, drought stress treatment was carried out at the seedling stage and a control group was set up. Seven indexes, namely relative plant fresh weight, reduction rate of relative water content, reduction rate of specific leaf weight, injury rate, reduction rate of water loss rate of detached leaves, increase rate of malondialdehyde content, and increase rate of proline content, of the 5-leaf stage plants of each Chinese cabbage germplasm material were measured. The membership function values and average membership function values of each index were calculated, and the drought tolerance was divided into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function values.

[0135] S301: First, polyethylene glycol (PEG-6000) with a molecular weight of 6000 Da was used to simulate drought stress. Specifically, a polyethylene glycol solution with a concentration of 10% was prepared, and the solution concentration was the mass-to-volume ratio (w / v). Then, for 100 Chinese cabbage germplasm materials, seeds of uniform size, plumpness, and health were selected for each material and sown in a 32-hole plug tray. When the seedlings reached the 5-leaf stage, the plants with consistent growth and good condition of each material were taken out with the substrate and placed in a 100 mL beaker. After standing for 12 h until the water in the substrate was exhausted, 80 mL of a 10% polyethylene glycol solution was poured into the beaker. After placing it at room temperature for 24 h, the 5-leaf stage plants of these 100 Chinese cabbage germplasm materials were subjected to index measurement; a control group was set for each material. The difference between the control group and the drought stress group was that the polyethylene glycol solution in the beaker was replaced with distilled water, and other experimental conditions were the same.

[0136] S302: Measure the relative plant fresh weight of the 5-leaf stage plants of each Chinese cabbage germplasm material. The calculation formula for the relative plant fresh weight is:

[0137] Relative plant fresh weight = fresh weight of the plant after drought stress treatment at the seedling stage / fresh weight of the plant in the control group;

[0138] In the formula: the units of the fresh weight of the plant after drought stress treatment at the seedling stage and the fresh weight of the plant in the control group are both g.

[0139] S303: Measure the relative water content reduction of the 5-leaf stage plants of each Chinese cabbage germplasm material. The calculation formula for the relative water content reduction is:

[0140] Relative water content reduction = (relative water content after drought stress treatment at the seedling stage - relative water content in the control group) / relative water content in the control group × 100%;

[0141] Among them, the calculation formula for the relative water content is:

[0142] Relative water content = (fresh weight of the leaf - dry weight of the leaf) / (fully turgid weight of the leaf - dry weight of the leaf);

[0143] In the formula: the units of the fresh weight of the leaf, the fully turgid weight of the leaf, and the dry weight of the leaf are all g.

[0144] The specific weighing method for the fresh weight of the leaf, the fully turgid weight of the leaf, and the dry weight of the leaf is as follows: Take 1 g of the 4th leaf of each plant, weigh the fresh weight of this leaf, then soak this leaf in distilled water until it reaches a constant weight, weigh the fully turgid weight of this leaf, and finally put this leaf into an oven and dry it at 80 °C until it reaches a constant weight, and weigh the dry weight of this leaf.

[0145] S304: Measure the decrease rate of specific leaf weight of the 5-leaf-stage plants of each Chinese cabbage germplasm material. The specific leaf weight is measured by the punching and weighing method. Avoid the main vein of the leaf and punch 20 small round pieces with a diameter of 0.5 cm, and then weigh them precisely. The calculation formula for the decrease rate of specific leaf weight is:

[0146] Decrease rate of specific leaf weight = (Specific leaf weight after seedling stage drought stress treatment - Specific leaf weight of the control group) / Specific leaf weight of the control group × 100%;

[0147] Among them, the calculation formula for the specific leaf weight is:

[0148] Specific leaf weight = Leaf weight / Leaf area;

[0149] In the formula:

[0150] The unit of leaf weight is g;

[0151] The unit of leaf area is cm 2 .

[0152] S305: Measure the injury rate of the 5-leaf-stage plants of each Chinese cabbage germplasm material. The calculation formula for the injury rate is:

[0153] Injury rate = (Conductivity value after seedling stage drought stress treatment - Conductivity value of the control group) / (Boiled conductivity value after seedling stage drought stress treatment - Conductivity value of the control group) × 100%;

[0154] The specific measurement methods for the conductivity value and the boiled conductivity value are as follows: Avoid the main vein of the leaf and punch 20 small round pieces with a diameter of 0.5 cm, add 5 mL of double-distilled water, soak for 5 h, and then measure the conductivity value; then perform a boiling water bath for 15 min, and measure the boiled conductivity after cooling to room temperature. The units of both the conductivity value and the boiled conductivity value are mS / cm.

[0155] S306: Measure the decrease rate of the water loss rate of detached leaves of the 5-leaf-stage plants of each Chinese cabbage germplasm material. The calculation formula for the decrease rate of the water loss rate of detached leaves is:

[0156] Decrease rate of water loss rate of detached leaves = (Water loss rate of detached leaves after seedling stage drought stress treatment - Water loss rate of detached leaves of the control group) / Water loss rate of detached leaves of the control group × 100%;

[0157] Among them, the water loss rate of detached leaves = (FW1 - FW2) / DW / (t1 - t2);

[0158] In the formula:

[0159] The unit of the water loss rate of detached leaves is mg·g -1 ·min -1 ;

[0160] FW1 and FW2 are the fresh weights at the first and second weighings respectively, with the unit of g;

[0161] DW is the dry weight of the leaf, with the unit of g;

[0162] t1 - t2 is the time difference between the first and second weighings, with the unit of min;

[0163] The specific measurement methods for the fresh weights at the first and second weighings and the dry weight of the leaf are as follows: Take leaves from the same part, accurately weigh the fresh weight, which is the fresh weight at the first weighing. Then lay the leaves flat on the wire mesh and let them air dry naturally in a laboratory with constant temperature, no wind, and no direct sunlight. After 2 hours, weigh the air-dried weight, which is the fresh weight at the second weighing. Then dry the leaves in an oven at 80°C until they reach a constant weight, and weigh the dry weight of the leaves.

[0164] S307: Measure the increase rate of malondialdehyde content in the 5-leaf-stage plants of each Chinese cabbage germplasm material. The calculation formula for the increase rate of malondialdehyde content is:

[0165] Increase rate of malondialdehyde content = (Malondialdehyde content after seedling stage drought stress treatment - Malondialdehyde content of the control group) / Malondialdehyde content of the control group × 100%;

[0166] The specific measurement method for the malondialdehyde content is as follows:

[0167] (1) Sample treatment

[0168] Use the malondialdehyde (MDA) content detection kit from Beijing Solarbio Science & Technology Co., Ltd., with the product number BC0020. According to the instruction manual, add 0.1 g of leaf tissue to 1 mL of extraction solution, homogenize in an ice bath and place it in a 1.5 mL centrifuge tube. Set the relative centrifugal force to 8000 g and centrifuge at 4°C for 10 min. Take the supernatant and place it on ice for further measurement;

[0169] (2) Measurement steps

[0170] Measurement tube: 200 μL of sample + 600 μL of MDA detection working solution + 200 μL of reagent three;

[0171] Blank tube: 200 μL of distilled water + 600 μL of MDA detection working solution + 200 μL of reagent three;

[0172] Place the above mixed solution in a 100°C water bath for 60 min and then cool it in an ice bath. Set the relative centrifugal force to 10000 g and centrifuge at room temperature for 10 min. Preheat the spectrophotometer for more than 30 min, zero it with distilled water, take the supernatant into a 1 mL glass cuvette, and measure the absorbance of each sample at 532 nm and 600 nm;

[0173] (3) Calculation of malondialdehyde content

[0174] ΔA532 = A532 测定 - A532 空白 ;

[0175] In the formula:

[0176] ΔA532 represents the difference in absorbance measured at a wavelength of 532 nm;

[0177] A532 测定 refers to the absorbance of the sample solution in the measurement tube at a wavelength of 532 nm;

[0178] A532 空白 refers to the absorbance of the blank tube solution at a wavelength of 532 nm;

[0179] ΔA600 = A600 测定 - A600 空白 ;

[0180] In the formula:

[0181] ΔA600 represents the difference in absorbance measured at a wavelength of 600 nm;

[0182] A600 测定 refers to the absorbance of the sample solution in the measurement tube at a wavelength of 600 nm;

[0183] A600 空白 refers to the absorbance of the blank tube solution at a wavelength of 600 nm;

[0184] ΔA = ΔA532 - ΔA600;

[0185] In the formula:

[0186] ΔA is the corrected difference in absorbance, which corrects the absorbance reading at 532 nm by subtracting the background absorption at 600 nm to eliminate non - specific absorbance interference;

[0187] MDA content = [ΔA × V 反总 / (ε × d) × 10 9 / (W × V 样本 / V 提取 ) × F = 32.258 × ΔA / W × F;

[0188] In the formula:

[0189] The unit of MDA content is nmol / g;

[0190] ΔA is the corrected difference in absorbance;

[0191] V 反总 is the total reaction volume, which is 1 mL in this experiment;

[0192] ε is the molar absorptivity;

[0193] d is the optical path length of the glass cuvette, in cm;

[0194] 10 9 is the unit conversion factor to convert the result from mol / L to nmol / mL;

[0195] W is the fresh weight of the sample, i.e., the weight of the leaf tissue used for MDA extraction, in g;

[0196] V 样本 is the volume of the sample added to the reaction system, which is 200 μL in this experiment;

[0197] V 提取 is the total volume of the extraction solution, which is 1 mL in this experiment;

[0198] F is the dilution factor. If the sample is not diluted, then F = 1.

[0199] S308: Determine the increase in proline content of the 5-leaf stage plants of each Chinese cabbage germplasm material. The calculation formula for the increase in proline content is:

[0200] Increase in proline content = (Proline content after drought stress treatment at the seedling stage - Proline content of the control group) / Proline content of the control group × 100%;

[0201] The specific determination method for the proline content is as follows:

[0202] (1) Sample treatment

[0203] Use the proline (Pro) content detection kit from Beijing Solarbio Science & Technology Co., Ltd., product number BC0290. Operate according to the instructions. Take 0.1 g of leaf tissue, add 1 mL of extraction solution, homogenize in an ice bath, place it in a 1.5 mL centrifuge tube, extract by boiling water bath oscillation for 10 min, set the relative centrifugal force to 10000 g and centrifuge at room temperature for 10 min, and take the supernatant for measurement after cooling;

[0204] (2) Measurement steps

[0205] Treatment of the standard product: Take 10 mg of proline and add 1 mL of distilled water to prepare a 10 mg / mL standard product, and dilute it with distilled water to 40 μg / mL, 20 μg / mL, 10 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL respectively;

[0206] Add the following reagents to a 1.5 mL centrifuge tube, mix well, keep warm in a boiling water bath for 30 min, shake once every 10 min, and measure after cooling;

[0207] Standard tube: 500 μL standard + 500 μL Reagent 1 + 500 μL Reagent 2;

[0208] Assay tube: 500 μL supernatant + 500 μL Reagent 1 + 500 μL Reagent 2;

[0209] Blank tube: 500 μL distilled water + 500 μL Reagent 1 + 500 μL Reagent 2;

[0210] Preheat the spectrophotometer for more than 30 min and zero it with distilled water. Transfer the supernatant to a 1 mL cuvette and measure the absorbance of each sample at 520 nm;

[0211] (3) Calculation of proline content

[0212] ΔA = A 测定管 - A 空白管 ;

[0213] In the formula:

[0214] ΔA is the corrected absorbance difference;

[0215] A 测定管 is the absorbance of the sample solution in the assay tube at a wavelength of 520 nm;

[0216] A 空白管 is the absorbance of the blank tube solution at a wavelength of 520 nm;

[0217] ΔA 标准 = A 标准管 - A 空白管 ;

[0218] In the formula:

[0219] ΔA 标准 is the difference between the absorbance of the standard tube solution and the absorbance of the blank tube solution;

[0220] A 标准管 is the absorbance of the standard tube solution at a wavelength of 520 nm;

[0221] A 空白管 is the absorbance of the blank tube solution at a wavelength of 520 nm;

[0222] Using the standard solution concentration as the abscissa and ΔA 标准 as the ordinate, plot the standard curve to obtain the linear regression equation y = kx + b, and substitute ΔA into the equation to get x;

[0223] Pro content = x × V 提 / W = x / W;

[0224] In the formula:

[0225] The content of Pro is the content of proline in the sample, with the unit of μg / g;

[0226] x is the concentration of proline in the sample solution calculated from the standard curve regression equation, with the unit of μg / mL;

[0227] V 提 is the total volume of the extraction solution, which is 1 mL in this experiment, that is, the volume of the extraction solution added after homogenizing the sample;

[0228] W is the fresh weight of the sample, that is, the weight of the leaf tissue used for extracting proline, with the unit of g.

[0229] S309: Calculate the membership function values and average membership function values of each index, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value.

[0230] The calculation method of the membership function value is as follows:

[0231] For the indexes positively correlated with drought tolerance, that is, relative plant fresh weight, the formula U(X i )=(X i -X min ) / (X max -X min ) is adopted;

[0232] For the indexes negatively correlated with drought tolerance, that is, relative water content decline rate, specific leaf weight decline rate, injury rate, in vitro leaf water loss rate decline rate, malondialdehyde content increase rate, proline content increase rate, the formula U(X i )=1-(X i -X min ) / (X max -X min ) is adopted;

[0233] In the formula:

[0234] U(X i ) is the membership function value of this index in the i-th Chinese cabbage germplasm material to be identified;

[0235] X i is the measured value of this index of the i-th Chinese cabbage germplasm material to be identified, and i is B1, B2, B3... B100;

[0236] X max is the maximum value of the measured values of this index among the 100 Chinese cabbage germplasm materials to be identified;

[0237] X min is the minimum value of the measured values of this index among the 100 Chinese cabbage germplasm materials to be identified;

[0238] The calculation method of the average membership function value is as follows:

[0239]

[0240] In the formula:

[0241] is the average membership function value of the 7 indicators of the i-th Chinese cabbage germplasm material to be identified;

[0242] n = 7;

[0243] The described X i 、X max 、X min all have the same unit;

[0244] The classification standard of the drought tolerance is as follows:

[0245] Level 1 is drought-tolerant, that is

[0246] Level 2 is relatively drought-tolerant, that is

[0247] Level 3 is moderately drought-tolerant, that is

[0248] Level 4 is drought-sensitive, that is

[0249] Level 5 is extremely drought-sensitive, that is

[0250] The results of the seven indicators of relative plant fresh weight, relative water content reduction rate, specific leaf weight reduction rate, injury rate, in vitro leaf water loss rate reduction rate, malondialdehyde content increase rate and proline content increase rate measured in steps S302 to S309 and the average membership function values of these seven indicators are shown in Table 2, and the seedling stage drought tolerance of these 100 Chinese cabbage germplasm materials is divided into five levels.

[0251] Level 1, drought-tolerant materials: B19, B20, B25, B26, B27, B28, B31, B33, B37, B39, B41, B49, B51, B59, B73, B85, B91, B94, B96, a total of 19, accounting for 19%;

[0252] Level 2, relatively drought-tolerant materials: B2, B9, B10, B11, B12, B13, B16, B17, B21, B29, B30, B32, B34, B35, B36, B38, B4, B40, B42, B44, B46, B47, B48, B50, B52, B53, B54, B55, B56, B57, B61, B62, B67, B69, B7, B71, B74, B75, B76, B77, B78, B79, B80, B81, B82, B88, B89, B90, B98, B99, B100, a total of 51, accounting for 51%;

[0253] Level 3, moderately drought-tolerant materials: B1, B3, B5, B6, B8, B14, B18, B22, B23, B24, B43, B45, B58, B64, B66, B72, B83, B84, B87, B92, B93, B95, a total of 22, accounting for 22%;

[0254] Level 4, drought-sensitive materials: B15, B60, B65, B68, B70, B86, a total of 6, accounting for 6%;

[0255] Level 5, extremely drought-sensitive materials: B63, B97, a total of 2, accounting for 2%.

[0256] Table 2 Membership function values and comprehensive evaluation table of drought tolerance indexes of 100 Chinese cabbage germplasm materials at the seedling stage

[0257]

[0258]

[0259]

[0260] S4: Comprehensive identification: Screen Chinese cabbage germplasm materials with drought tolerance identification results of level 1 at both the germination stage and the seedling stage, and determine them as drought-tolerant germplasm materials.

[0261] As can be seen from Table 3, among the 100 Chinese cabbage germplasm materials tested, the drought tolerance identification results of 22 materials are consistent at the germination stage and the seedling stage. B19, B28, B33, B49, B59, B94 are at level 1 and belong to drought-tolerant materials; B46, B62, B100 are at level 2 and belong to relatively drought-tolerant materials; B6, B8, B23, B45, B58, B66, B92, B95 are at level 3 and belong to moderately drought-tolerant materials; B15, B60, B65, B68, B86 are at level 4 and belong to drought-sensitive materials; B63, B97 are at level 5 and belong to extremely drought-sensitive materials.

[0262] Taking the identification results at the germination stage and the seedling stage both being level 1 as the screening criteria for drought-tolerant germplasm materials, the drought-tolerant germplasm materials among these 100 Chinese cabbage germplasm materials can be screened out as B19, B28, B33, B49, B59, and B94.

[0263] Table 3 Comparison table of drought tolerance grading of 100 Chinese cabbage germplasm materials at the germination stage and the seedling stage

[0264]

[0265]

[0266] In the specific implementation process, in step S201, 10 seeds of uniform size, plump and healthy are selected from each material and placed in a petri dish with a diameter of 9 cm lined with double filter papers. 10 mL of polyethylene glycol solution with a solution concentration of 15% is measured with a measuring cylinder and poured into the petri dish respectively. After covering the petri dish lid, it is continuously cultured in an environment with a temperature of 25 °C, 12 h of light and 12 h of darkness for 8 d. A control group is set for each material. The difference between the control group and the experimental group subjected to drought stress is that the polyethylene glycol solution in the petri dish is replaced with distilled water, and other experimental conditions are the same. The above treatment steps are set with 3 repetitions; in step S301, seeds of uniform size, plump and healthy are selected from each material and sown in a 32-hole plug tray. When the seedlings reach the 5-leaf stage, the plants with consistent growth and good condition of each material are taken out together with the substrate by the roots and placed in a 100 mL beaker. After standing for 12 h to deplete the water in the substrate, 80 mL of polyethylene glycol solution with a solution concentration of 10% is poured into the beaker. After placing it at room temperature for 24 h, the index measurement is carried out on the 5-leaf stage plants of these 100 Chinese cabbage germplasm materials. A control group is set for each material. The difference between the control group and the drought stress group is that the polyethylene glycol solution in the beaker is replaced with distilled water, and other experimental conditions are the same. The above treatment steps are set with 3 repetitions.

[0267] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A method for identifying drought-tolerant germplasm materials of Chinese cabbage, characterized in that: It includes the following steps: S1: Prepare Chinese cabbage germplasm materials to be identified: Select seeds of different Chinese cabbage germplasm materials and 5-leaf-stage plants corresponding to these Chinese cabbage germplasm materials; S2: Germination stage identification: Perform drought stress treatment on the seeds of each Chinese cabbage germplasm material during the germination stage and set a control group. Measure four indicators, namely relative root length, relative seedling height, germination drought resistance index, and relative vigor index, of the seeds of each Chinese cabbage germplasm material. Calculate the membership function values and average membership function values of each indicator, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value; S3: Seedling stage identification: Perform drought stress treatment on the 5-leaf-stage plants of each Chinese cabbage germplasm material during the seedling stage and set a control group. Measure seven indicators, namely relative plant fresh weight, relative water content reduction, specific leaf weight reduction, injury rate, reduction rate of in vitro leaf water loss rate, increase rate of malondialdehyde content, and increase rate of proline content, of the 5-leaf-stage plants of each Chinese cabbage germplasm material. Calculate the membership function values and average membership function values of each indicator, and classify the drought tolerance into five levels: level 1, level 2, level 3, level 4, and level 5 according to the average membership function value; S4: Comprehensive identification: Screen the Chinese cabbage germplasm materials with the identification results of level 1 in both the germination stage and the seedling stage, and determine them as drought-tolerant germplasm materials.

2. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S2, the drought stress treatment during the germination stage includes: Place the seeds of each Chinese cabbage germplasm material in a petri dish containing 10 mL of polyethylene glycol solution with a solution concentration of 15%, cover the petri dish lid, and continuously culture them in an environment with a temperature of 25°C, 12 h of light and 12 h of darkness for 8 days. Observe and measure the germination of these seeds every day, and consider the seeds showing white tips as germination; The molecular weight of the polyethylene glycol is 6000 Da; The solution concentration is the mass-to-volume ratio.

3. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S2, the calculation formula for the relative root length is: Relative root length = root length after drought stress treatment during the germination stage / root length of the control group × 100%; The calculation formula for the relative seedling height is: Relative seedling height = seedling height after drought stress treatment during the germination stage / seedling height of the control group × 100%.

4. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 2, characterized in that: In step S2, the calculation formula for the germination drought resistance index is: Germination drought resistance index = seed germination index after drought stress treatment during the germination stage / seed germination index of the control group; Among them, the calculation formula for the seed germination index is: Seed germination index = 1.00×Rd2 + 0.75×Rd4 + 0.50×Rd6 + 0.25×Rd8; In the formula: Rd2, Rd4, Rd6, and Rd8 are the germination rates on the 2nd, 4th, 6th, and 8th days respectively; 1.00, 0.75, 0.50, and 0.25 are the drought resistance coefficients assigned to the corresponding germination days; The calculation formula for the relative vigor index is: Relative vigor index = vigor index after drought stress treatment during the germination stage / vigor index of the control group × 100%; Among them, the calculation formula for the vigor index is: Vigor index = radicle length × germination index; Among them, the calculation formula for the germination index is: In the formula: Gi is the number of germinations on the i-th day; Di is the corresponding germination day, that is, Di = i; n is the number of test days, that is, n = 8.

5. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S3, the seedling stage drought stress treatment includes: putting the 5-leaf stage plants of each Chinese cabbage germplasm material together with the substrate into a beaker, standing for 12 h, pouring 80 mL of polyethylene glycol solution with a solution concentration of 10% into the beaker, and placing it at room temperature for 24 h; the molecular weight of the polyethylene glycol is 6000 Da; the solution concentration is the mass-volume ratio.

6. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S3, the calculation formula for the relative plant fresh weight is: Relative plant fresh weight = fresh weight of the plant after the seedling stage drought stress treatment / fresh weight of the plant in the control group; The calculation formula for the reduction rate of relative water content is: Reduction rate of relative water content = (relative water content after the seedling stage drought stress treatment - relative water content in the control group) / relative water content in the control group × 100%; Among them, the calculation formula for the relative water content is: Relative water content = (fresh weight of the leaf - dry weight of the leaf) / (saturated water weight of the leaf - dry weight of the leaf).

7. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S3, the calculation formula for the reduction rate of specific leaf weight is: Reduction rate of specific leaf weight = (specific leaf weight after the seedling stage drought stress treatment - specific leaf weight in the control group) / specific leaf weight in the control group × 100%; Among them, the calculation formula for the specific leaf weight is: Specific leaf weight = leaf weight / leaf area; The calculation formula for the injury rate is: Injury rate = (conductivity value after the seedling stage drought stress treatment - conductivity value in the control group) / (boiled conductivity value after the seedling stage drought stress treatment - conductivity value in the control group) × 100%.

8. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: In step S3, the calculation formula for the reduction rate of the water loss rate of detached leaves is: Reduction rate of the water loss rate of detached leaves = (water loss rate of detached leaves after the seedling stage drought stress treatment - water loss rate of detached leaves in the control group) / water loss rate of detached leaves in the control group × 100%; The calculation formula for the increase rate of malondialdehyde content is: Increase rate of malondialdehyde content = (malondialdehyde content after the seedling stage drought stress treatment - malondialdehyde content in the control group) / malondialdehyde content in the control group × 100%; The calculation formula for the increase rate of proline content is: Increase rate of proline content = (proline content after the seedling stage drought stress treatment - proline content in the control group) / proline content in the control group × 100%.

9. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: The calculation method for the membership function value is: For the indicators that are positively correlated with drought tolerance, namely relative root length, relative seedling height, germination drought resistance index, relative vigor index, and relative plant fresh weight, the formula U(X i )=(X i -X min ) / (X max -X min ) is used; For the indexes negatively correlated with drought tolerance, namely the decline rate of relative water content, the decline rate of specific leaf weight, the injury rate, the decline rate of water loss rate of detached leaves, the increase rate of malondialdehyde content, and the increase rate of proline content, the formula U(X i ) = 1 - (X i - X min ) / (X max - X min ) is used; In the formula: U(X i ) is the membership function value of this index in the i-th Chinese cabbage germplasm material to be identified; X i is the measured value of this index for the i-th Chinese cabbage germplasm material to be identified, where i corresponds to the number of different Chinese cabbage germplasm materials; X max is the maximum value among the measured values of this index for all Chinese cabbage germplasm materials to be identified; X min is the minimum value among the measured values of this index for all Chinese cabbage germplasm materials to be identified; The calculation method for the average membership function value is: In the formula: is the average membership function value of n indicators of the i-th Chinese cabbage germplasm material to be identified; n is the number of measured index items.

10. The identification method of a drought-tolerant germplasm material of Chinese cabbage according to claim 1, characterized in that: The grading standard for drought tolerance is: Level 1 is drought-tolerant, i.e., Level 2 indicates relatively drought-tolerant, that is Level 3 indicates medium drought tolerance, that is Level 4 is drought-sensitive, i.e., Level 5 is extremely sensitive to drought, i.e., Among them is the average membership function value of n indicators of the i-th Chinese cabbage germplasm material to be identified.

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