Method for identifying low-nitrogen resistance of corn inbred line in seedling stage

Abstract

The invention belongs to the technical field of corn breeding, and particularly relates to a method for identifying low-nitrogen resistance of a corn inbred line in a seedling stage. The identification method comprises the following steps: respectively carrying out low nitrogen stress treatment and normal nitrogen supply treatment on corn inbred line seedlings to obtain low nitrogen stress treatment character values and normal nitrogen supply treatment character values of a plurality of indexes; wherein the plurality of indexes comprise data values of root system characters, dry weight and root-shoot ratio; obtaining low-nitrogen-resistant coefficients corresponding to a plurality of indexes of the corn inbred line seedlings; performing principal component analysis to obtain a low-nitrogen resistance comprehensive index of each principal component and a membership function value and a contribution rate corresponding to the low-nitrogen resistance comprehensive index; solving a low-nitrogen resistance comprehensive index weight of each principal component; the low-nitrogen-resistant comprehensive evaluation value of each corn inbred line seedling is obtained, the low-nitrogen-resistant characteristic of the corn inbred line seedling stage is evaluated according to the low-nitrogen-resistant comprehensive evaluation value, and then germplasm resources high in low-nitrogen-resistant capacity are screened out.

Description

Technical Field

[0001] The invention belongs to the technical field of corn breeding, and in particular relates to a method for identifying the low-nitrogen tolerance characteristics of corn inbred lines at the seedling stage. Background Art

[0002] As a food crop, corn has been widely used in feed, food, industry and commercial circulation, and plays a vital role in ensuring my country's food security. Nitrogen is an important nutrient that affects corn production, development, yield and quality. In order to increase corn yield, excessive nitrogen application often occurs in agricultural production, resulting in nitrogen fertilizer application far higher than the actual demand, resulting in reduced nitrogen fertilizer utilization, increased production costs and environmental pollution. Studies have shown that there are obvious differences in nitrogen utilization efficiency among different genotypes of corn germplasm resources, which provides the possibility for breeding nitrogen-efficient corn varieties. However, the breeding of low-nitrogen tolerant varieties requires corresponding corn inbred lines as parents. Therefore, identifying and screening low-nitrogen tolerant corn inbred lines, breeding new nitrogen-efficient corn varieties, and improving the nitrogen fertilizer utilization efficiency of corn are effective ways to achieve green and healthy development of the corn industry.

[0003] The identification and screening period of corn tolerance to low nitrogen is mainly divided into the seedling stage and the whole growth period. Previous studies have shown that the nitrogen utilization rate of corn in the early stage is crucial to increasing corn yield in the later stage. In addition, compared with the identification during the whole growth period, the identification during the seedling stage has the advantages of short time, small environmental impact and strong experimental repeatability, and has been widely used in the study of corn low nitrogen stress. Related studies analyzed multiple main trait indicators of 26 different genotypes of corn parents at the seedling stage and showed that the coefficient of variation of total dry weight per plant changed the most, which was extremely significantly negatively correlated with the number of leaves with nitrogen deficiency symptoms and significantly positively correlated with nitrogen conversion rate, and three low nitrogen resistant germplasms were screened out. Related studies conducted low nitrogen stress studies on 51 corn hybrids at the seedling stage and found that low nitrogen stress had a significant effect on 13 morphological and physiological indicators of each corn hybrid at the seedling stage, but there were large genotype differences between hybrids. After comprehensive evaluation using principal component analysis and membership function analysis, three low nitrogen resistant corn varieties such as Zhenghong 311 were screened out. Other studies have shown that compared with low-nitrogen sensitive corn varieties, low-nitrogen tolerant corn varieties have a lower decline in biomass, root system and other indicators during the seedling stage. They can maintain root vitality under low nitrogen stress, promote root water absorption and transport of nutrients, control the increase in root canal ratio, and maintain the growth and development of the aboveground part. The above studies show that corn's tolerance to low nitrogen is a comprehensive trait, and a single indicator cannot fully reflect the plant's response to low nitrogen stress, so a comprehensive evaluation of multiple indicators is needed.

[0004] From the above, we can see that the current identification indicators for corn's tolerance to low nitrogen are not unified, resulting in low accuracy of the identification results. Summary of the invention

[0005] In order to solve the problem that the identification indicators of corn low nitrogen tolerance in the prior art are not unified, resulting in low accuracy of identification results, the present invention adopts a hydroponic test method to analyze the differences in biomass, root characteristics and other seedling traits of 108 corn inbred lines under normal nitrogen and low nitrogen conditions. By calculating the low nitrogen tolerance coefficient of each inbred line and conducting multivariate statistical analysis based on this coefficient, it is intended to identify and screen out corn inbred lines that show good low nitrogen tolerance. The present invention will provide strong technical support and material basis for the genetic research of corn low nitrogen tolerance, and will also help to breed new low nitrogen tolerant corn varieties with excellent comprehensive traits. To achieve the above purpose, the present invention adopts the following technical scheme:

[0006] The present invention provides a method for identifying the low nitrogen tolerance characteristics of corn inbred lines at the seedling stage, comprising the following steps:

[0007] Maize inbred line seedlings were subjected to low nitrogen stress treatment and normal nitrogen supply treatment, respectively, to obtain trait values ​​of multiple indicators under low nitrogen stress treatment and normal nitrogen supply treatment; wherein the multiple indicators were root- and biomass-related indicators, including data values ​​of root traits, dry weight and root-to-crown ratio.

[0008] The low nitrogen tolerance coefficient corresponding to multiple indicators of the corn inbred line seedlings is obtained according to the ratio of the low nitrogen stress treatment trait value to the normal nitrogen supply treatment trait value.

[0009] The low nitrogen tolerance coefficients corresponding to the multiple indicators of the corn inbred line seedlings are respectively subjected to principal component analysis to obtain a comprehensive index of low nitrogen tolerance of each principal component and its corresponding membership function value and contribution rate.

[0010] According to the contribution rate, the weight of the comprehensive index of low nitrogen tolerance of each principal component in all comprehensive indexes of low nitrogen tolerance is calculated, and the weight of the comprehensive index of low nitrogen tolerance of each principal component is obtained.

[0011] According to the product of the weight of the comprehensive index of low nitrogen tolerance of each principal component and the corresponding membership function value, the comprehensive evaluation value of low nitrogen tolerance of each corn inbred line seedling is obtained, and the low nitrogen tolerance characteristics of the corn inbred line seedling are evaluated according to the comprehensive evaluation value of low nitrogen tolerance. Among them, the larger the comprehensive evaluation value of low nitrogen tolerance, the stronger the low nitrogen tolerance of the corn inbred line seedling.

[0012] The invention provides a method for identifying low nitrogen tolerance characteristics of corn inbred lines at the seedling stage, including unified identification indicators, by measuring multiple indicators related to roots and biomass of the corn inbred lines to be tested, obtaining low nitrogen tolerance coefficients corresponding to multiple indicators of the corn inbred line seedlings, and then using the low nitrogen tolerance coefficients to perform principal component analysis, and finally obtaining a low nitrogen tolerance comprehensive evaluation value, and the corn inbred lines with low nitrogen tolerance characteristics can be identified and screened according to the low nitrogen tolerance comprehensive evaluation value. The identification result obtained by the method for identifying low nitrogen tolerance characteristics of corn inbred lines at the seedling stage provided by the invention has high accuracy, and can accurately evaluate the low nitrogen tolerance of corn germplasm resources.

[0013] Among them, the related traits of root traits, dry weight and root-to-crown ratio are all traits related to roots and biomass. The present invention selects the data values ​​of the related traits of root traits, dry weight and root-to-crown ratio as the identification indicators in the above identification method, which not only ensures the unification of the identification indicators for the low-nitrogen tolerance of corn, but also improves the accuracy of the identification results of the low-nitrogen tolerance of corn, and further can more accurately screen out germplasm resources with strong low-nitrogen tolerance.

[0014] The root-to-shoot ratio refers to the ratio of the dry weight of the underground part of a plant to the dry weight of the aboveground part. This ratio reflects the correlation between the growth of the underground and aboveground parts of a plant and is an important indicator of the biological characteristics of a plant.

[0015] The identification method provided by the present invention is a study on the low nitrogen tolerance of corn inbred lines. Therefore, selecting the data values ​​of the above-mentioned root traits, dry weight and root-to-shoot ratio as identification indicators can directly reflect the good low nitrogen tolerance characteristics of the corn inbred lines. It is a comprehensive trait indicator joint identification, which can ensure a high accuracy of the identification results.

[0016] Preferably, the root traits include total root length, root surface area, average root diameter, total root volume, number of root tips, and number of root branches.

[0017] Among them, root surface area: This refers to the total surface area of ​​all roots in the plant's root system. The surface area of ​​the root system is crucial for plants to absorb water and nutrients, because a larger surface area means more absorption sites.

[0018] Total root volume: This refers to the total volume occupied by all the roots of a plant's root system. It reflects the space occupied by the roots in the soil and their growth conditions.

[0019] Total root length: This refers to the total length of all roots of the plant root system. Total root length is an important indicator to measure the growth of the root system, which reflects the extension and exploration ability of the root system in the soil.

[0020] Root diameter: This usually refers to the average diameter of the root system or the diameter of a specific root. Root diameter is related to the root system's support capacity and the efficiency of water and nutrient transfer.

[0021] Average root diameter: This refers to the average diameter of all roots in the root system. It provides an overall description of the thickness of the root system and helps to understand the growth status and physiological characteristics of the root system.

[0022] Root tip number: This refers to the number of all root tips in the root system. The root tip is the most active part of the root system and is responsible for cell division and elongation, so the root tip number can reflect the growth potential and vitality of the root system.

[0023] Root branch number: This refers to the number of all branches in the root system. The branching ability of the root system is crucial to its distribution and stability in the soil. The more branches there are, the wider the root system is distributed in the soil and the better its stability.

[0024] Preferably, the dry weight includes above-ground dry weight, underground dry weight and total dry weight.

[0025] Wherein, the method for determining the dry weight of the aboveground part is: weighing the aboveground part by a drying method.

[0026] The method for determining the dry weight of the underground part is: weighing the underground part by a drying method.

[0027] The total dry weight is the sum of the above-ground dry weight and the underground dry weight.

[0028] Preferably, the calculation formula for the low nitrogen tolerance coefficient corresponding to each index of the corn inbred line is as follows:

[0029] Low nitrogen tolerance coefficient = low nitrogen stress treatment trait value / normal nitrogen supply treatment trait value.

[0030] Among them, the smaller the low nitrogen tolerance coefficient, the more sensitive the trait corresponding to this indicator is to low nitrogen treatment, and the larger the low nitrogen tolerance coefficient, the less sensitive the trait corresponding to this indicator is to low nitrogen treatment. When the low nitrogen tolerance coefficients of multiple trait values ​​of an inbred line are all greater than the set value, it indicates that the inbred line has a strong ability to tolerate low nitrogen.

[0031] Preferably, the calculation formula of the membership function value is as follows:

[0032]

[0033] Where, j = 1, 2, 3, ..., n; μ(X j ) represents the membership function value of the comprehensive index of low nitrogen tolerance of the jth principal component; the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of each principal component is measured in parallel 3 to 4 times, X j represents the average value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jminrepresents the minimum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmax It represents the maximum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel measurements.

[0034]

[0035] Where, j = 1, 2, 3, ..., n; W j represents the weight of the comprehensive index of low nitrogen tolerance of the jth principal component in all comprehensive indexes of low nitrogen tolerance; P j It represents the contribution rate of the jth principal component of each cross line to the comprehensive index of low nitrogen tolerance.

[0036]

[0037] Wherein, j = 1, 2, 3, ..., n; D represents the comprehensive evaluation value of low nitrogen tolerance of each cross line under low nitrogen stress conditions.

[0038] The present invention adopts an indoor hydroponic method to evaluate the low nitrogen tolerance of the test corn inbred lines at the seedling stage, and selects a total of 10 indicators including underground dry weight, aboveground dry weight, total dry weight, root-to-shoot ratio and 6 root traits for measurement, and simultaneously adopts a low nitrogen tolerance coefficient (indicator low nitrogen stress / indicator normal nitrogen) for analysis, which can not only reflect the level of each indicator in response to low nitrogen stress, but also reduce the influence caused by environmental factors and nutrients.

[0039] Preferably, the method further includes clustering the comprehensive evaluation value of low nitrogen tolerance to obtain four groups: strong low nitrogen tolerance type, low nitrogen tolerance type, intermediate type, and low nitrogen sensitive type, and the low nitrogen tolerance ability represented by the four groups decreases in sequence.

[0040] The present invention uses cluster analysis to divide 108 maize inbred lines into four groups: strong low-nitrogen tolerance type, low-nitrogen tolerance type, intermediate type, and low-nitrogen sensitive type. Among them, there are 7 maize inbred lines with strong low-nitrogen tolerance and 25 maize inbred lines with low-nitrogen tolerance. These inbred lines can be applied to breeding practice to improve the nitrogen utilization rate of maize germplasm resources.

[0041] Preferably, principal component analysis is performed to obtain a comprehensive index of low nitrogen tolerance for each principal component, which is as follows: principal component analysis is performed on the low nitrogen tolerance coefficients corresponding to multiple indicators of the corn inbred line seedlings using SPSS software, and the original data of the low nitrogen tolerance coefficients corresponding to root traits, dry weight and root-to-crown ratio are subjected to dimensionality reduction processing to obtain 1 to 4 independent comprehensive indexes of low nitrogen tolerance.

[0042] Under normal nitrogen and low nitrogen stress levels, the low nitrogen tolerance coefficients of the indicators of total root length, root tip number, root branch number, underground dry weight and root-crown ratio can be used as the main indicators for evaluating the low nitrogen tolerance of corn inbred lines at the seedling stage. The present invention has experimentally studied that under normal nitrogen and low nitrogen stress levels, the low nitrogen tolerance coefficients of the indicators of total root length, root tip number, root branch number, underground dry weight and root-crown ratio can be used as the main indicators for evaluating the low nitrogen tolerance of corn inbred lines at the seedling stage. The present invention provides that the low nitrogen tolerance characteristics of corn inbred lines at the seedling stage can be identified by using the above indicators, and the identification results have a high accuracy.

[0043] Preferably, the corn inbred line seedlings are cultured under conditions where the nitrogen content is 0.03 mmol / L to 0.06 mmol / L for 14 to 16 days.

[0044] Preferably, the corn seedlings to be tested are obtained after processing inbred corn kernels.

[0045] The treatment method is as follows: soak the inbred corn kernels and germinate them in a dark environment at 28°C; when the radicle grows to about 1.0 cm to 2.0 cm, select the uniformly growing seedlings, place them in a moist environment, and continue to culture them in a nutrient solution containing 1 / 2 Hoagland to obtain the corn seedlings to be tested.

[0046] The formula composition of the 1 / 2 Hoagland nutrient solution is the improved Hoagland nutrient solution purchased by Beijing Coolaibo Technology Co., Ltd.

[0047] Wherein, the corn seedlings to be tested are corn seedlings in the one-leaf-one-heart stage.

[0048] The soaking time of the inbred corn kernels is 12 hours.

[0049] The seedlings are cultured in a nutrient solution containing 1 / 2 Hoagland.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The present invention provides a method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage, and the identification method is a study on the low nitrogen tolerance of corn inbred lines. The identification result obtained by the method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage provided by the present invention has a high accuracy and can accurately evaluate the low nitrogen tolerance of corn germplasm resources.

[0052] 2. In order to find out the identification method and index of corn inbred lines resistant to low nitrogen at seedling stage, and screen out corn inbred lines with strong low nitrogen tolerance and applicable to breeding practice, 108 corn inbred lines were used as materials, and hydroponic experiments were carried out at seedling stage under normal nitrogen and low nitrogen level treatments to measure 10 indexes related to roots and biomass. Correlation analysis, principal component analysis, cluster analysis and regression analysis were performed using the low nitrogen tolerance coefficient to identify and screen corn inbred lines with low nitrogen tolerance.

[0053] The above results showed that different nitrogen treatments had significant effects on various indicators of maize inbred lines at the seedling stage, but the degree of influence was different between inbred lines and indicators. The principal component analysis converted 10 individual indicators into 3 comprehensive indicators. The tested maize inbred lines were divided into 4 types through cluster analysis based on the membership function value and the comprehensive evaluation value of low nitrogen tolerance of inbred lines, including 7 strong low nitrogen tolerance types, 25 low nitrogen tolerance types, 52 intermediate types, and 24 low nitrogen sensitive types. Stepwise regression analysis showed that the low nitrogen tolerance coefficients of total root length, root tip number, root branch number, underground dry weight, and root-to-crown ratio can be used as the main indicators for identifying the low nitrogen tolerance characteristics of maize inbred lines at the seedling stage.

[0054] 3. The research results of the present invention show that under normal nitrogen and low nitrogen stress levels, the low nitrogen tolerance coefficients of total root length, root tip number, root branch number, underground dry weight and root-crown ratio can be used as the main indicators for evaluating the low nitrogen tolerance of maize inbred lines at the seedling stage. The principal component analysis method based on the membership function value screened out 11 low nitrogen tolerant maize inbred lines such as FBHJ, LH224 and Xi14 from 108 maize inbred lines; and 24 low nitrogen sensitive maize inbred lines such as PHN11, PHR25 and PHR63. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a correlation analysis of the low nitrogen tolerance coefficient of corn in the seedling stage of the present invention, wherein TRL is total root length; TRSA is root surface area; RAD: average root diameter; RV is total root volume; RT is the number of root tips; RF is the number of root branches; RDW: underground dry weight; SDW is the aboveground dry weight; TDW: total dry weight; RSR is root-to-crown ratio.

[0056] Figure 2 This is a cluster diagram of the low nitrogen tolerance coefficient of corn at the seedling stage in the present invention.

[0057] Figure 3 It is a flow chart of the present invention. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from relevant scientific research units or commercial channels.

[0059] See also Figure 3 A method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage comprises the following steps:

[0060] S1. The corn inbred lines planted in the nursery box are subjected to low nitrogen stress treatment and normal nitrogen supply treatment respectively to obtain the trait values ​​of the low nitrogen stress treatment and the trait values ​​of the normal nitrogen supply treatment of multiple indicators; wherein the multiple indicators are indicators related to roots and biomass, including data values ​​of root traits, dry weight and root-to-crown ratio.

[0061] Among them, root traits include total root length, root surface area, average root diameter, total root volume, number of root tips, and number of root branches.

[0062] The method for determining the dry weight of the aboveground part is: weighing the aboveground part using the drying method.

[0063] S2. Obtaining the low nitrogen tolerance coefficient corresponding to multiple indicators of the corn inbred line according to the ratio of the low nitrogen stress treatment trait value to the normal nitrogen supply treatment trait value.

[0064] Among them, the calculation formula for the low nitrogen tolerance coefficient corresponding to each index of corn inbred line is as follows:

[0065] Low nitrogen tolerance coefficient = low nitrogen stress treatment trait value / normal nitrogen supply treatment trait value.

[0066] Among them, the smaller the low nitrogen tolerance coefficient, the more sensitive the trait corresponding to this indicator is to low nitrogen treatment, and the larger the low nitrogen tolerance coefficient, the less sensitive the trait corresponding to this indicator is to low nitrogen treatment. When the low nitrogen tolerance coefficients of multiple trait values ​​of an inbred line are all greater than the set value, it indicates that the inbred line has a strong ability to tolerate low nitrogen.

[0067] The above indicators are the data values ​​of low nitrogen tolerance coefficient corresponding to total root length, root surface area, average root diameter, total root volume, number of root tips, number of root branches, underground dry weight, aboveground dry weight, total dry weight and root-crown ratio.

[0068] S3. Performing principal component analysis on the low nitrogen tolerance coefficients corresponding to the multiple indicators of the corn inbred line seedlings, respectively, to obtain a comprehensive indicator of low nitrogen tolerance of each principal component and its corresponding membership function value and contribution rate.

[0069] The calculation formula of the membership function value is as follows:

[0070]

[0071] Where, j = 1, 2, 3, ..., n; μ(X j ) represents the membership function value of the comprehensive index of low nitrogen tolerance of the jth principal component; the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of each principal component is measured in parallel 3 to 4 times, X j represents the average value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmin represents the minimum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmax It represents the maximum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel measurements.

[0072] S4. Calculate the weight of the comprehensive index of low nitrogen tolerance of each principal component in all comprehensive indexes of low nitrogen tolerance according to the contribution rate, and obtain the weight of the comprehensive index of low nitrogen tolerance of each principal component.

[0073] in,

[0074] Where, j = 1, 2, 3, ..., n; W j represents the importance of the low nitrogen tolerance comprehensive index of the jth principal component in all the low nitrogen tolerance comprehensive indexes, i.e., its weight; j It represents the contribution rate of the jth principal component of each cross line to the comprehensive index of low nitrogen tolerance.

[0075] S5. According to the product of the weight of the comprehensive index of low nitrogen tolerance of each principal component and the corresponding membership function value, the comprehensive evaluation value of low nitrogen tolerance of each corn inbred line seedling is obtained, and the low nitrogen tolerance characteristics of the corn inbred line seedling stage are evaluated according to the comprehensive evaluation value of low nitrogen tolerance. Among them, the larger the comprehensive evaluation value of low nitrogen tolerance, the stronger the low nitrogen tolerance of the corn inbred line.

[0076] in,

[0077] Wherein, j = 1, 2, 3, ..., n; D represents the comprehensive evaluation value of low nitrogen tolerance of each cross line under low nitrogen stress conditions.

[0078] Furthermore, it also includes clustering analysis of the comprehensive evaluation values ​​of low nitrogen tolerance to obtain four groups: strong low nitrogen tolerance type, low nitrogen tolerance type, intermediate type, and low nitrogen sensitive type. The low nitrogen tolerance ability represented by the four groups decreases in sequence.

[0079] Furthermore, cluster analysis was used to divide the 108 maize inbred lines into four groups: strong low-nitrogen tolerance, low-nitrogen tolerance, intermediate, and low-nitrogen sensitive. Among them, there were 7 maize inbred lines with strong low-nitrogen tolerance and 25 maize inbred lines with low-nitrogen tolerance. These inbred lines can be applied to breeding practice to improve the nitrogen utilization rate of maize germplasm resources.

[0080] Furthermore, principal component analysis is performed to obtain a comprehensive index of low nitrogen tolerance for each principal component: principal component analysis is performed on the low nitrogen tolerance coefficients corresponding to multiple indicators of the corn inbred line seedlings using SPSS software, and the original data of the low nitrogen tolerance coefficients corresponding to root traits, dry weight and root-to-crown ratio are subjected to dimensionality reduction processing to obtain 1 to 4 independent comprehensive indexes of low nitrogen tolerance.

[0081] Example 1: A method for identifying low nitrogen tolerance characteristics of corn inbred lines at the seedling stage

[0082] This example provides a method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage. The low nitrogen tolerance of 108 corn inbred lines at the seedling stage was identified by a hydroponic test method to evaluate the low nitrogen tolerance of these inbred lines.

[0083] 1. Test corn materials

[0084] The information of the 108 maize inbred lines tested is shown in Table 1, all of which were provided by the Maize Research Institute of Jilin Academy of Agricultural Sciences.

[0085] Table 1 Names and numbers of tested maize inbred lines

[0086] serial number Inbred line serial number Inbred line serial number Inbred line serial number Inbred line 1 FBJ 28 LH217 55 Ji818 82 Ji D008 2 IBB15 29 LH200 56 Ji 803 83 Ji D284 3 IBC2 30 LH184 57 Ji V057 84 Ji V665 4 PHK29 31 PHAW6 58 444 85 Ji V0665 5 PHR25 32 PHEM9 59 Ji K287 86 Ji V6266 6 PHV78 33 PHFA5 60 Summary 31 87 Jilin A318 7 PHK42 34 PHHB9 61 Dan 340 88 WC009 8 PHK76 35 PHKE6 62 Department 14 89 JIA388 9 PHN11 36 PHMK0 63 Ji921 90 Jilin A372 10 PHV63 37 PHRE1 64 Ji 842 91 A5147 11 PHP02 38 PHW53 65 K10 92 Ji A239 12 PHR47 39 PHW80 66 Zheng 58 93 JiDH8216 13 PHW52 40 PHEG9 67 B73 94 Ji D060 14 PHN82 41 PHAA0 68 PH6WC 95 Ji D043 15 PHP55 42 PHTD5 69 Ji 854 96 Ji D69105 16 PHJ89 43 PHKM5 70 IV-144 97 Ji D7480 17 PHJ90 44 PHR63 71 Cheng 18 98 Ji D1863 18 PHN66 45 LH284 72 Dan598 99 Ji D224 19 PHT11 46 Ji V417 73 Ji V022 100 Ji D254 20 PHBW8 47 PH4CV 74 Changqi-2 101 Ji DH96 21 PHGV6 48 Mo17 75 IV-287 102 Ji V203 22 PHN18 49 Ji1037 76 JiLM16 103 Ji V2416 23 PHR31 50 Total 344 77 Ji A71 104 Ji V044 24 PHT69 51 Ji992 78 Ji A5001 105 Ji K390 25 LH172 52 J001 79 Ji DH9090 106 Ji V2943 26 LH224 53 Ji846 80 Ji D165 107 Ji V2962 27 LH166 54 Ji 63 81 Ji D65377 108 Ji V5239

[0087] 2. Methods

[0088] 1. Experimental design

[0089] The experiment was conducted in the artificial climate chamber of the Corn Research Institute of Jilin Academy of Agricultural Sciences in May 2023. The nutrient solution preparation method refers to the improved formula of nutrient solution for normal nitrogen supply and low nitrogen treatment reported by Wu Yawei et al. The specific steps are as follows:

[0090] S1. Seed selection: Select seeds with clean and uniform surface.

[0091] S2. Seed treatment: First disinfect with 10% hydrogen peroxide for 30 minutes, wash with distilled water 4 times and soak for 12 hours, then transfer to a germination tray covered with moistened filter paper and germinate at 28°C in the dark.

[0092] S3. Cultivation of maize inbred line seedlings: When the radicle grows to about 1.0 cm, select the uniformly growing seedlings and place them in a double layer of moist filter paper, roll up the filter paper with the seedlings and place it vertically in a seedling box containing 1 / 2 Hoagland nutrient solution, and continue to cultivate in an artificial climate chamber. The cultivation conditions are: temperature 26°C, humidity 72%.

[0093] 2. Treatment of corn inbred seedlings

[0094] Two treatment groups were set up, namely the low nitrogen stress treatment group and the normal nitrogen supply treatment group.

[0095] When the above-mentioned corn inbred seedlings have one leaf and one heart, select the corn inbred seedlings with the same growth, remove the endosperm and randomly divide them into two groups: a low nitrogen stress treatment group and a normal nitrogen supply treatment group. Among them, the corn inbred seedlings in the low nitrogen stress treatment group were treated with low nitrogen stress; the corn inbred seedlings in the normal nitrogen supply treatment group were treated with normal nitrogen supply. The specific treatment methods are as follows:

[0096] The normal nitrogen supply treatment group was transplanted into a seedling box containing 1 / 2 Hoagland nutrient solution with normal nitrogen level for cultivation.

[0097] The low nitrogen stress treatment group was transplanted into a seedling box containing 1 / 2 Hoagland nutrient solution with low nitrogen level for cultivation.

[0098] Each treatment was repeated three times, with 8 seedlings in each repeat. The system was aerated with an air pump for 8 h every day, and the nutrient solution containing 1 / 2 Hoagland was replaced every 3 days.

[0099] Among them, 1 / 2 Hoagland nutrient solution is the improved Hoagland nutrient solution purchased by Beijing Coolaibo Technology Co., Ltd.

[0100] Normal nitrogen level refers to a nitrogen concentration of 4mmol / L, NN.

[0101] Low nitrogen level refers to a nitrogen concentration of 0.04mmol / L, LN.

[0102] 3. Obtaining the trait values ​​of multiple indicators under low nitrogen stress treatment and normal nitrogen supply treatment

[0103] After the corn inbred line seedlings in the low nitrogen stress treatment group and the normal nitrogen supply treatment group were treated with 1 / 2 Hoagland nutrient solution for 15 days, the low nitrogen tolerance traits of multiple indicators corresponding to the corn inbred line seedlings in the low nitrogen stress treatment group and the normal nitrogen supply treatment group were measured, and the low nitrogen stress treatment trait values ​​and normal nitrogen supply treatment trait values ​​of the multiple indicators were obtained, respectively.

[0104] Among them, multiple indicators include data values ​​of root traits, dry weight and root-to-shoot ratio.

[0105] Root traits included total root length, root surface area, average root diameter, total root volume, number of root tips and number of root branches.

[0106] The dry weight includes the aboveground dry weight, underground dry weight and total dry weight.

[0107] Three seedlings were randomly selected for each replicate to measure the index. The specific measurement method is as follows:

[0108] (1) Root traits: Root surface area, total root volume, total root length, root diameter, average root diameter, number of root tips, number of root branches, etc. were measured using a root scanner (Epson Expression 1000xl, WinRHIZO).

[0109] Among them, root surface area: This refers to the total surface area of ​​all roots in the plant's root system. The surface area of ​​the root system is crucial for plants to absorb water and nutrients, because a larger surface area means more absorption sites.

[0110] Total root volume: This refers to the total volume occupied by all the roots of a plant's root system. It reflects the space occupied by the roots in the soil and their growth conditions.

[0111] Total root length: This refers to the total length of all roots of the plant root system. Total root length is an important indicator to measure the growth of the root system, which reflects the extension and exploration ability of the root system in the soil.

[0112] Root diameter: This usually refers to the average diameter of the root system or the diameter of a specific root. Root diameter is related to the root system's support capacity and the efficiency of water and nutrient transfer.

[0113] Average root diameter: This refers to the average diameter of all roots in the root system. It provides an overall description of the thickness of the root system and helps to understand the growth status and physiological characteristics of the root system.

[0114] Root tip number: This refers to the number of all root tips in the root system. The root tip is the most active part of the root system and is responsible for cell division and elongation, so the root tip number can reflect the growth potential and vitality of the root system.

[0115] Root branch number: This refers to the number of all branches in the root system. The branching ability of the root system is crucial to its distribution and stability in the soil. The more branches there are, the wider the root system is distributed in the soil and the better its stability.

[0116] (2) Determination of dry weight of aboveground part: The aboveground part of corn plants was placed in a kraft paper bag and dried in an oven at 110°C for 20 min. The aboveground part was then dried at 60°C until constant weight was reached and weighed.

[0117] (3) Determination of dry weight of underground part: The underground part of corn plants was placed in a kraft paper bag and dried in an oven at 110°C for 20 min. The dried part was then weighed after being dried at 60°C until constant weight was reached.

[0118] (4) Total dry weight: The total dry weight is calculated by adding the aboveground dry weight and the underground dry weight.

[0119] (5) Root-to-crown ratio: The root-to-crown ratio refers to the ratio of the dry weight of the underground part to the aboveground part of the plant. This ratio reflects the correlation between the growth of the underground part and the aboveground part of the plant and is an important indicator of the biological characteristics of the plant. The root-to-crown ratio is calculated by the underground dry weight / aboveground dry weight.

[0120] 4. Achieving tolerance to low nitrogen coefficient

[0121] The index data measured in the low nitrogen stress treatment group and the normal nitrogen supply treatment group were statistically analyzed to calculate the low nitrogen tolerance coefficient of multiple measured indicators of maize inbred seedlings. Among them, the multiple measured indicators are total root length, root surface area, average root diameter, total root volume, number of root tips, number of root branches, underground dry weight, aboveground dry weight, total dry weight and root-crown ratio.

[0122]

[0123] 5. Principal component analysis, calculate the comprehensive index of low nitrogen tolerance and its corresponding membership function value and contribution rate

[0124] The above 10 low nitrogen tolerance coefficients were statistically analyzed by the principal component analysis module of the DPS software, and the membership function value and contribution rate of the comprehensive index of low nitrogen tolerance of each principal component were calculated using formula (1). The contribution rate means that the evaluation results of the 10 low nitrogen tolerance coefficients are defined as 100%, but since the 10 low nitrogen tolerance coefficients are correlated, the key can be used to combine the correlated indicators together to form a comprehensive index, simplifying the 10 indicators into 3 comprehensive indicators, and then the comprehensive index is the percentage of the evaluation results of the 10 low nitrogen tolerance coefficients, which is the contribution rate of this comprehensive index.

[0125]

[0126] Wherein, j = 1, 2, 3, ..., n, n is a positive integer; μ(X j ) represents the membership function value of the comprehensive index of low nitrogen tolerance of the jth principal component; the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of each principal component is measured in parallel 3 to 4 times, X j represents the average value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jminrepresents the minimum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmax It represents the maximum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel measurements.

[0127] 6. Obtaining the weight of the comprehensive index of low nitrogen tolerance

[0128] Formula (2) is used to obtain the weight of the comprehensive index of low nitrogen tolerance of each principal component according to the weight of the comprehensive index of low nitrogen tolerance of each principal component in all comprehensive indexes of low nitrogen tolerance.

[0129]

[0130] Wherein, j = 1, 2, 3, ..., n, n is a positive integer; W j represents the importance of the comprehensive index of low nitrogen tolerance of the jth principal component in all comprehensive indexes of low nitrogen tolerance, i.e., its weight; j It represents the contribution rate of the jth principal component of each cross line to the comprehensive index of low nitrogen tolerance.

[0131] 7. Obtaining the comprehensive evaluation value of low nitrogen tolerance

[0132] Formula (3) was used to obtain the comprehensive evaluation values ​​of low nitrogen tolerance of different maize inbred lines, and the comprehensive evaluation values ​​of low nitrogen tolerance were ranked; the larger the value of the comprehensive evaluation value of low nitrogen tolerance, the stronger the low nitrogen tolerance of the maize inbred line.

[0133]

[0134] Wherein, j=1, 2, 3, ..., n, n is a positive integer; D represents the comprehensive evaluation value of low nitrogen tolerance of each cross line under low nitrogen stress conditions.

[0135] 8. Data Processing

[0136] Microsoft Excel 2007 was used for data processing and drawing, and IBM SPSS22.0 software was used for principal component analysis and cluster analysis to evaluate the low nitrogen tolerance of maize inbred lines.

[0137] Specifically, the method used in the data processing and analysis method is as follows:

[0138] Microsoft Office Excel 2013 was used for data processing and drawing, and DPS19.05 IBM SPSS Statistics 21.0 software was used to organize the data analysis of low nitrogen tolerance indexes at the seedling stage. The DPS data processing system was used for significance analysis, and the R language was used to draw cluster diagrams. The comprehensive evaluation method based on membership function was used to evaluate the low nitrogen tolerance of the tested maize inbred line seedlings.

[0139] 3. The test and identification results are as follows:

[0140] 1. Differences in traits among maize inbred lines under low nitrogen stress

[0141] A hydroponic experiment was conducted on 108 maize inbred lines. The trait test results of the maize inbred line seedlings in the normal nitrogen supply treatment group and the low nitrogen treatment group were statistically analyzed, and the low nitrogen tolerance coefficient corresponding to each index was calculated. The results are shown in Table 2.

[0142] Table 2 Differences in seedling traits of maize inbred lines under two nitrogen treatments

[0143]

[0144] From the results in Table 2, we can see that 10 indicators, including total root length, root surface area, average root diameter, total root volume, number of root tips, number of root branches, underground dry weight, aboveground dry weight, total dry weight and root-to-crown ratio, all showed changes to varying degrees.

[0145] The seedling traits of the maize inbred lines in the low nitrogen treatment group were mainly manifested in a significant decrease in the number of root tips, the number of root branches, and the aboveground dry weight; while the root-shoot ratio increased significantly. The low nitrogen tolerance coefficient can be used to preliminarily identify the low nitrogen tolerance of the tested maize inbred lines. The seven indicators of total root length, root surface area, total root volume, number of root tips, number of root branches, aboveground dry weight, and total dry weight were all less than 1, indicating that low nitrogen treatment affects the growth and development of maize.

[0146] Under different nitrogen treatments, all indicators of maize inbred seedlings showed certain variation, and the degree of variation of each indicator was quite different. The coefficients of variation of the 10 indicators under two nitrogen treatments and the low nitrogen tolerance coefficient were all greater than 18%, indicating that there were large genotypic differences in the low nitrogen tolerance of different maize inbred seedlings. At the same time, since the size of the low nitrogen tolerance coefficient can not only reflect the difference of indicators under different nitrogen treatments, but also eliminate the errors and biological differences between inbred lines, it is more accurate to use the low nitrogen tolerance coefficient for subsequent analysis.

[0147] 2. Differences in traits among maize inbred lines under low nitrogen stress in maize inbred line seedlings

[0148] The correlation results of the low nitrogen tolerance coefficients of the 10 indicators measured are as follows: Figure 1 shown.

[0149] Figure 1 The results of the analysis showed that the low nitrogen tolerance coefficients of underground dry weight, aboveground dry weight, and total dry weight were positively correlated with the low nitrogen tolerance coefficients of root traits such as total root length, root surface area, average root diameter, total root volume, number of root tips, and number of root branches. This indicates that the greater the low nitrogen tolerance coefficient of dry matter under low nitrogen treatment, the greater the low nitrogen tolerance coefficient of root traits; that is, the greater the change in root traits under low nitrogen treatment, the greater the relative change in dry matter. The above results show that these 10 indicators can be used as indicators for screening low nitrogen tolerant inbred lines of corn, but there are significant or extremely significant differences in the correlation between multiple indicators, and the low nitrogen tolerance information provided has a certain degree of repeatability.

[0150] 3. Principal component analysis of low nitrogen tolerance measurement indicators of maize inbred line seedlings

[0151] The tolerance of corn to low nitrogen is affected by many factors, and the correlation of the tolerance coefficients of various indicators is obviously different. The tolerance coefficient of a single indicator cannot accurately evaluate the tolerance of corn inbred line seedlings to low nitrogen, so a comprehensive evaluation of the tolerance coefficient of inbred lines is needed.

[0152] The present invention uses SPSS software to perform principal component analysis on the low nitrogen tolerance coefficients of 10 indicators of 108 corn inbred lines, and according to the principle that the characteristic value is greater than 1, the original 10 indicators are converted into 3 independent comprehensive indicators. The results of the principal component analysis are shown in Table 3.

[0153] Table 3 Principal component analysis of low nitrogen tolerance coefficients of maize inbred lines seedlings

[0154] index Principal component 1 Principal component 2 Principal component 3 Total root length <![CDATA[0.763]]> <![CDATA[-0.318]]> <![CDATA[0.246]]> Root surface area <![CDATA[0.922]]> <![CDATA[0.145]]> <![CDATA[0.136]]> Average root diameter <![CDATA[-0.083]]> <![CDATA[0.924]]> <![CDATA[0.083]]> Total root volume <![CDATA[0.768]]> <![CDATA[0.491]]> <![CDATA[0.138]]> Number of root tips <![CDATA[0.641]]> <![CDATA[-0.559]]> <![CDATA[0.283]]> Number of root branches <![CDATA[0.825]]> <![CDATA[-0.321]]> <![CDATA[0.114]]> Dry weight of underground part <![CDATA[0.842]]> <![CDATA[0.223]]> <![CDATA[0.109]]> Above ground dry weight <![CDATA[0.829]]> <![CDATA[0.154]]> <![CDATA[-0.455]]> Total dry weight <![CDATA[0.884]]> <![CDATA[0.167]]> <![CDATA[-0.32]]> Root-shoot ratio <![CDATA[-0.135]]> <![CDATA[0.236]]> <![CDATA[0.885]]> Eigenvalue <![CDATA[5.317]]> <![CDATA[1.79]]> <![CDATA[1.302]]> Contribution rate (%) <![CDATA[ 53 .174]]> <![CDATA[ 17 .902]]> <![CDATA[ 13 .023]]> Cumulative contribution rate (%) <![CDATA[ 53 .174]]> <![CDATA[ 71 .077]]> <![CDATA[ 84 .099]]>

[0155] The results in Table 3 show that the contribution rates of the three principal components are 53.174%, 17.902% and 18.793% respectively, and the cumulative contribution rate is 84.099%, which represents most of the information of low nitrogen tolerance indicators of maize inbred seedlings. Among them, the first principal component has a larger eigenvector for total root length, root surface area, total root volume, number of root tips, number of root branches, underground dry weight, aboveground dry weight and total dry weight; the second principal component has a larger eigenvector for average root diameter; the third principal component has a larger eigenvector for root-crown ratio.

[0156] 4. Comprehensive evaluation value of low nitrogen tolerance of corn inbred line seedlings

[0157] The present invention uses (Formula 1) to calculate the membership function value of the comprehensive index of low nitrogen tolerance at the seedling stage of each main component, and calculates its weight (Formula 2) according to the contribution rate of each comprehensive index, so as to obtain the comprehensive evaluation value of low nitrogen tolerance of the test material (Formula 3), and the results are shown in Table 4.

[0158] Table 4 Analysis of low nitrogen tolerance coefficients and comprehensive evaluation values ​​of low nitrogen tolerance of various indicators in low nitrogen tolerant and low nitrogen sensitive maize inbred lines seedlings

[0159]

[0160] Note: The comprehensive evaluation value of low nitrogen tolerance is also called D value. The larger the D value, the stronger the comprehensive low nitrogen tolerance of the test material in the seedling stage.

[0161] The results in Table 4 show that the D value of the tested materials ranged from 0.173 to 0.755, with a mean and standard deviation of 0.378 and 0.110, respectively, and a coefficient of variation of 30.00%. Taking the D value as the standard for identifying the tolerance of maize inbred line seedlings to low nitrogen, the comprehensive evaluation values ​​of low nitrogen tolerance of 5 maize inbred lines, including FBHJ, LH224, Xi14, PHT69 and JiD284, were higher, indicating that these maize inbred line seedlings had strong tolerance to low nitrogen; the comprehensive evaluation values ​​of low nitrogen tolerance of 5 maize inbred line seedlings, including PHK76, Dan598, PHN11, PHR25 and PHR63, were lower, indicating that these maize inbred line seedlings had weak tolerance to low nitrogen.

[0162] 5. Cluster analysis of comprehensive evaluation values ​​of low nitrogen tolerance at the seedling stage

[0163] Cluster analysis was performed on 108 maize inbred lines based on the comprehensive evaluation value of low nitrogen tolerance, and the results are shown in Table 5.

[0164] Table 57 low nitrogen tolerance coefficients and comprehensive evaluation indicators of 7 varieties with strong low nitrogen tolerance

[0165]

[0166] Note: The comprehensive evaluation value of low nitrogen tolerance is also called D value. The larger the D value, the stronger the comprehensive low nitrogen tolerance of the test material in the seedling stage.

[0167] The results in Table 5 show that the tested maize inbred lines were divided into four groups: strong low nitrogen tolerance, low nitrogen tolerance, intermediate, and low nitrogen sensitive. The mean D values ​​were 0.672, 0.453, 0.339, and 0.254, respectively, and the standard deviations were 0.063, 0.045, 0.030, and 0.025, respectively. There were significant differences in D values ​​among the groups. Category I includes 7 maize inbred lines, such as FBHJ, LH224 and Xi14, which are strong low-nitrogen tolerant maize inbred lines; Category II includes 25 maize inbred lines, such as PHEM9, PHW52 and PHR31, which are low-nitrogen tolerant maize inbred lines; Category III includes 52 maize inbred lines, such as LH172, PHAW6 and PHN18, which are intermediate maize inbred lines; Category IV includes 24 maize inbred lines, such as JiA388, J001 and Ji803, which are low-nitrogen sensitive maize inbred lines, as shown in Tables 6 and Figure 2 shown.

[0168] Table 6 Low nitrogen tolerance coefficient and comprehensive evaluation value of low nitrogen tolerance of different groups

[0169]

[0170] Note: Different capital letters in the same column indicate significant at the P < 0.01 level, and different lowercase letters indicate significant at the P < 0.05 level.

[0171] 6. Regression analysis and identification and screening of low nitrogen tolerance indicators

[0172] According to the comprehensive evaluation value of low nitrogen tolerance of maize inbred seedlings (D), the low nitrogen tolerance coefficient (X) of each index was subjected to stepwise regression analysis, and a digital model for predicting low nitrogen tolerance was established to screen indicators closely related to the comprehensive evaluation value of low nitrogen tolerance, thereby simplifying the identification workload. With the comprehensive evaluation value of low nitrogen tolerance as the dependent variable and the low nitrogen tolerance coefficient of each index as the independent variable, a regression equation for identifying the low nitrogen tolerance of maize inbred seedlings was established: D = -0.054 + 0.024X total root length + 0.108X average root diameter + 0.008 root tip number + 0.020 root branch number + 0.033 underground dry weight + 0.018 root-crown ratio. The obtained regression equation determination coefficient R 2 =0.997, and the F test reached an extremely significant level, indicating that its prediction accuracy was high. According to the regression equation, the low nitrogen tolerance coefficients of total root length, root tip number, root branch number, underground dry weight and root-shoot ratio can be used as the main indicators for identifying the low nitrogen tolerance of maize inbred lines at the seedling stage.

[0173] The above experimental results show that under normal nitrogen and low nitrogen stress levels, the low nitrogen tolerance coefficients of total root length, root tip number, root branch number, underground dry weight and root-shoot ratio can be used as the main indicators to evaluate the low nitrogen tolerance of maize inbred lines at the seedling stage. The principal component analysis method based on the membership function value was used to screen out 11 low nitrogen tolerant maize inbred lines such as FBHJ, LH224 and Xi14 from 108 maize inbred lines; and 24 low nitrogen sensitive maize inbred lines such as PHN11, PHR25 and PHR63.

[0174] Nitrogen is one of the most absorbed nutrients in corn growth, and it is also a key element necessary for the synthesis of corn organic matter and endogenous metabolites. Therefore, conducting research on corn tolerance to low nitrogen is of great significance for improving the breeding of low nitrogen tolerant corn varieties, the efficiency of corn nitrogen fertilizer utilization, reducing the use of chemical fertilizers and protecting the ecological environment. At present, many studies have been done on the screening of corn tolerance to low nitrogen. The screening indicators include morphological indicators such as plant height, biomass, root morphology and yield, as well as physiological and biochemical indicators such as photosynthetic rate, nitrogen content and related enzyme activity. Xu Shiying et al. showed that the corn root system is the first organ to respond to low nitrogen stress, and nitrogen is preferentially accumulated in the root system, resulting in an increase in the root-crown ratio. It is believed that the number of lateral roots, the number of root tips and the root-crown ratio are greatly affected by genetic factors; Luo Yanhong believes that seven indicators such as single plant leaf area, root volume, and root-crown ratio can be used to evaluate or screen the ability of corn to tolerate low nitrogen in the seedling stage; Chun Liang et al. believe that the root dry weight, total root length and lateral root length in the corn root system traits are the main traits that respond to low nitrogen stress. However, there is no unified identification method that can be intuitively evaluated, and most of the research focuses on corn hybrids, and there are few reports on the low nitrogen tolerance of corn inbred lines. In this study, the indoor hydroponic method was used to evaluate the low nitrogen tolerance of the tested corn inbred lines at the seedling stage. A total of 10 indicators including underground dry weight, aboveground dry weight, total dry weight, root-to-shoot ratio and six root traits were selected for determination. At the same time, the low nitrogen tolerance coefficient (indicator low nitrogen stress / indicator normal nitrogen) was used for analysis, which can not only reflect the level of each indicator in response to low nitrogen stress, but also reduce the impact of environmental factors and nutrients. Based on correlation analysis, principal component analysis and regression analysis, the low nitrogen tolerance index of corn inbred lines at the seedling stage was screened and identified. It is believed that the low nitrogen tolerance coefficient of total root length, root tip number, root branch number, underground dry weight and root-to-shoot ratio can be used as the main identification index. Cluster analysis was used to divide the 108 corn inbred lines into four groups: strong low nitrogen tolerance type, low nitrogen tolerance type, intermediate type and low nitrogen sensitive type. Among them, there are 7 highly resistant to low-nitrogen corn inbred lines and 25 resistant to low-nitrogen corn inbred lines. These inbred lines can be applied to breeding practice to improve the nitrogen utilization rate of corn germplasm resources.

[0175] The present invention provides a method for identifying the low nitrogen resistance of corn inbred lines at the seedling stage, and the identification method is a study on the low nitrogen resistance of corn inbred seedlings. The identification result obtained by the method for identifying the low nitrogen resistance of corn inbred lines at the seedling stage provided by the present invention has a high accuracy, and can accurately evaluate the low nitrogen resistance of corn germplasm resources. In order to find out the identification method and index of corn inbred lines resistant to low nitrogen at the seedling stage, the present invention screens out corn inbred lines with strong low nitrogen resistance and can be applied to breeding practice. 108 corn inbred seedlings are used as materials, and hydroponic tests are carried out at the seedling stage under normal nitrogen and low nitrogen level treatments to measure 10 indicators related to roots and biomass. The low nitrogen resistance coefficient is used to perform correlation analysis, principal component analysis, cluster analysis, regression analysis and other methods to identify and screen corn inbred lines with low nitrogen resistance.

[0176] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0177] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage, characterized in that: The steps include: The corn inbred line seedlings are subjected to low nitrogen stress treatment and normal nitrogen supply treatment respectively, and the trait values ​​of the low nitrogen stress treatment and the trait values ​​of the normal nitrogen supply treatment of multiple indicators are obtained; wherein the multiple indicators include data values ​​of root traits, dry weight and root-to-crown ratio; Obtaining low nitrogen tolerance coefficients corresponding to multiple indicators of the corn inbred line seedlings according to the ratio of the low nitrogen stress treatment trait value to the normal nitrogen supply treatment trait value; Conducting principal component analysis on the low nitrogen tolerance coefficients corresponding to the multiple indicators of the corn inbred line seedlings respectively, to obtain the comprehensive index of low nitrogen tolerance of each principal component and its corresponding membership function value and contribution rate; According to the contribution rate, the weight of each principal component's comprehensive index of low nitrogen tolerance in all comprehensive indexes of low nitrogen tolerance is calculated; According to the product of the weight of the comprehensive index of low nitrogen tolerance of each principal component and the corresponding membership function value, the comprehensive evaluation value of low nitrogen tolerance of each maize inbred line seedling is obtained, and the low nitrogen tolerance characteristics of maize inbred lines at the seedling stage are evaluated according to the comprehensive evaluation value of low nitrogen tolerance.

2. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The root traits include total root length, root surface area, average root diameter, total root volume, number of root tips and number of root branches.

3. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The dry weight includes the aboveground dry weight, the underground dry weight and the total dry weight.

4. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The multiple indicators include total root length, number of root tips, number of root branches, underground dry weight and root-to-crown ratio.

5. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The calculation formula of the membership function value is as follows: Where, j = 1, 2, 3, ..., n; μ(X j ) represents the membership function value of the comprehensive index of low nitrogen tolerance of the jth principal component; the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of each principal component is measured in parallel 3 to 4 times, X j represents the average value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmin represents the minimum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations, X jmax It indicates the maximum value of the low nitrogen tolerance coefficient corresponding to the comprehensive index of low nitrogen tolerance of the jth principal component in the results of 3 to 4 parallel determinations; Where W j represents the weight of the comprehensive index of low nitrogen tolerance of the jth principal component in all comprehensive indexes of low nitrogen tolerance; P j It represents the contribution rate of the comprehensive index of low nitrogen tolerance of the jth principal component of each maize inbred line seedling; Where D represents the comprehensive evaluation value of low nitrogen tolerance of each maize inbred line seedling under low nitrogen stress conditions.

6. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The method for performing principal component analysis to obtain the comprehensive index of low nitrogen tolerance of each principal component is as follows: principal component analysis is performed on the low nitrogen tolerance coefficients corresponding to multiple indicators of the corn inbred line seedlings using SPSS software, and the original data of the low nitrogen tolerance coefficients corresponding to root traits, dry weight and root-to-shoot ratio are subjected to dimensionality reduction processing to obtain 1 to 4 independent comprehensive indexes of low nitrogen tolerance.

7. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: The low nitrogen stress treatment conditions are: placing the corn inbred line seedlings under the condition of a nitrogen content of 0.03 mmol / L to 0.06 mmol / L and culturing them for 14 to 16 days.

8. The method for identifying the low nitrogen tolerance of corn inbred lines at the seedling stage according to claim 1, characterized in that: It also includes cluster analysis of the comprehensive evaluation values ​​of low nitrogen tolerance to obtain four groups: strong low nitrogen tolerance type, low nitrogen tolerance type, intermediate type, and low nitrogen sensitive type. The low nitrogen tolerance ability represented by the four groups decreases in sequence.

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