A method for evaluating peanut calcium deficiency tolerance based on pod fullness and its application in breeding.

Abstract

This invention relates to the fields of crop genetics, breeding, and cultivation physiology, specifically to a method for evaluating peanut stress tolerance, and more particularly to a method for evaluating peanut calcium deficiency tolerance based on pod fullness and its application in breeding. The method includes: planting the peanut variety to be tested under preset calcium deficiency stress conditions; after harvesting, measuring the pod kernel weight percentage and the full pod kernel weight percentage; calculating the pod fullness decay index (SDI); and screening calcium deficiency-tolerant peanut varieties based on the SDI values. This method overcomes the cumbersome and time-consuming nature of traditional calcium testing techniques, is suitable for early screening of large-scale breeding populations in the field, and can effectively identify and eliminate calcium-sensitive materials with severe "false fullness," significantly improving the breeding efficiency of calcium deficiency-tolerant peanut varieties. This invention also protects specific calcium deficiency-tolerant peanut varieties screened using this method and their application in calcium-deficient soils.

Description

Technical Field

[0001] This invention relates to the fields of crop genetics and breeding and cultivation physiology, specifically to a method for evaluating peanut stress tolerance, and more particularly to a method for evaluating peanut calcium deficiency tolerance based on pod fullness and its application in breeding. Background Technology

[0002] peanut( Arachis hypogaea Peanut kernels (L.) are rich in protein and fat, which helps alleviate the contradiction between oil and fat demand in my country. The growth, development, and quality of peanut kernels determine the yield and value of peanuts. As a cash crop with a high commercialization rate, peanuts are both edible and exportable, making them one of the main agricultural products promoting sustainable agricultural development.

[0003] Calcium is an essential nutrient element for plants, playing a crucial role throughout the entire plant growth and development process, from seed germination to vegetative growth, reproductive growth, and finally fruit ripening. Furthermore, calcium plays a vital role in signal regulation during plant growth and development. Calcium ions participate in the entire process from seed germination to flowering and fruiting, extensively regulating physiological activities. Calcium also acts as a second messenger within plant cells, translating external stimuli into regulations governing plant cell metabolic activities. Exogenous calcium application can promote peanut pod development through calcium-related proteins in signal transduction pathways, while providing sufficient nutrition to the above-ground pegs to facilitate their transition to the reproductive development stage.

[0004] Soil acidification leading to calcium deficiency has severely impacted peanut yield. Identifying and evaluating the sensitivity of major peanut varieties and germplasm resources to calcium deficiency, and screening for calcium-tolerant varieties, is crucial for peanut production in red soil drylands, calcium-tolerant breeding, and ensuring oilseed security. When peanuts are deficient in calcium, the plants and pods typically exhibit stunted growth, weak main stems, fewer branches, fewer fruits, fewer full pods, smaller and less plump pods, and an increase in rotten and empty pods. Furthermore, the fat and protein content in the kernels decreases, and the oleic acid / linoleic acid ratio drops. In drylands dominated by red soil, peanut yield reduction due to embryo abortion and empty kernels has been a long-standing problem.

[0005] Currently, there are two main types of methods for evaluating the calcium nutritional status or calcium deficiency tolerance of peanuts: The first is soil and plant chemical analysis, such as measuring the exchangeable calcium content in the soil and the calcium concentration in plant leaves or pegs. These methods require specialized equipment, are cumbersome to operate, and are costly. Furthermore, the relationship between plant calcium concentration and final pod fullness is not linear, making it difficult to directly reflect yield loss. The second method is indirect evaluation based on phenotypes, such as observing the rate of empty pods and the rate of double-kernel full pods. However, traditional empty pod statistics cannot distinguish between "false full pods" that appear plump but have atrophied kernels, which is precisely the key factor leading to hidden yield reduction due to calcium deficiency.

[0006] Therefore, there is an urgent need in this field for a phenotypic evaluation method that can directly, accurately, and quickly reflect the kernel filling ability of peanuts under calcium-deficient conditions and is applicable to large-scale breeding population screening. Summary of the Invention

[0007] The purpose of this invention is to provide a method for evaluating peanut calcium deficiency tolerance based on pod fullness and its application in breeding, thereby addressing the problems existing in the prior art. The method provided by this invention can directly and efficiently evaluate peanut calcium deficiency tolerance, and can also be used to rapidly screen calcium-tolerant germplasm resources, accelerating the peanut breeding process.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for evaluating the calcium deficiency tolerance of peanuts based on pod fullness, the method comprising the following steps: Peanuts to be tested were grown under calcium deficiency stress until maturity; Harvest the peanut pods to be tested, and determine the weight percentage of kernels in the pods and the weight percentage of kernels in the full pods, respectively; The pod fullness decay index of the peanut to be tested is calculated based on the weight percentage of the pod kernels and the weight percentage of the full kernels. The calcium deficiency tolerance of the tested peanut was evaluated based on the pod fullness decay index.

[0009] Optionally, the lower the pod fullness decay index of the peanut being tested, the stronger its tolerance to calcium deficiency.

[0010] Optionally, the calcium deficiency stress conditions include natural field conditions with exchangeable calcium content in the soil below 400 mg / kg or a controlled culture environment using a nutrient solution with a calcium concentration below 2.0 mmol / L.

[0011] Optionally, the formula for calculating the weight percentage of the pod kernels is as follows: ; Wherein, W1 is the weight percentage of pod kernels; M1 is the weight of pods in g; and M2 is the weight of shelled kernels in g.

[0012] Optionally, the formula for calculating the weight percentage of the full-bodied kernels is as follows: ; Where W2 is the weight percentage of full-bodied kernels; N1 is the weight of shelled kernels, in g; Alternatively, the formula for calculating the weight percentage of the full-bodied kernels is as follows: ; Wherein, W2 is the weight percentage of full pods; N0 is the weight of shelled kernels of full pods remaining after removing 20 pods per plant, in g; M2 is the weight of shelled kernels of 20 pods per plant, in g; and N2 is the weight of all full pods per plant, in g.

[0013] Optionally, the formula for calculating the pod fullness decay index is as follows: ; Wherein, SDI is the pod fullness decay index; W1 is the pod kernel weight percentage; and W2 is the full pod kernel weight percentage.

[0014] This invention provides the application of the above-described method in screening calcium-deficient peanut varieties.

[0015] This invention provides a method for screening calcium-deficient peanut varieties. The method includes calculating the pod fullness decay index of the peanut to be tested using the above-mentioned method, and comparing the obtained pod fullness decay index with a preset threshold T. If the pod fullness decay index is less than or equal to the preset threshold T, the peanut to be tested is determined to be a calcium-deficient peanut variety. The preset threshold T is in the range of 0.20-0.35.

[0016] Optionally, the preset threshold T is 0.25.

[0017] This invention provides the application of the above-described method in flowering breeding, wherein flowering breeding includes early screening of calcium-deficiency tolerant individual plants or lines from hybrid offspring populations, mutation populations, or naturally mutated populations.

[0018] This invention provides a method for breeding calcium-deficiency tolerant peanut varieties, comprising the following steps: hybridization using calcium-deficiency tolerant peanut germplasm as parents or creating a breeding population using mutagenesis technology; planting the breeding population under calcium deficiency stress conditions; screening plants with low pod fullness attenuation index using the above method; and conducting multi-generation propagation and trait stability identification on the screened plants to finally obtain new calcium-deficiency tolerant peanut varieties.

[0019] The present invention discloses the following technical effects: 1. Novel Indicators and Direct Mechanism: This invention is the first to propose the "Score Index (SDI)" as a core indicator. It accurately captures the key physiological defect of "false fullness" caused by calcium deficiency, which is directly linked to the final yield and has clear biological significance. Therefore, this invention reveals for the first time the phenomenon that the kernel fullness inside peanut "fullness" undergoes a specific decline under calcium deficiency stress, and based on this, establishes a rapid, intuitive phenotypic screening indicator directly related to the final yield.

[0020] 2. Simple operation and easy to promote: Only conventional harvesting, sorting, and weighing are required, without the need for complex and expensive chemical analysis instruments, making it suitable for large-scale implementation in breeding fields. Therefore, the method provided by this invention overcomes the cumbersome and delayed nature of traditional calcium testing techniques and is suitable for early screening of large-scale breeding populations in the field.

[0021] 3. Highly efficient screening with reliable results: This method can quickly identify and eliminate genotypes highly sensitive to calcium deficiency in early generations, concentrating resources on promising tolerant materials and significantly improving breeding efficiency. Therefore, the method provided by this invention can effectively identify and eliminate calcium-sensitive materials with severe "false pod filling," significantly improving the breeding efficiency of calcium-tolerant peanut varieties.

[0022] 4. High application value: The selected calcium-tolerant varieties can be directly used in agricultural production on calcium-deficient soils, reducing the amount of calcium fertilizer applied, lowering costs and environmental risks, and improving the production stability of peanuts on marginal land. Therefore, the method provided by this invention can screen specific calcium-tolerant peanut varieties, accelerating the peanut breeding process. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating the evaluation and screening method described in this invention; Figure 2 The chart shows the pod filling attenuation index (SDI) ranking of 32 peanut varieties in Example 1, illustrating the differences in calcium deficiency tolerance among the varieties. The horizontal axis represents the variety, and the vertical axis represents the SDI. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: A method for evaluating peanut calcium deficiency tolerance based on pod fullness A method for evaluating peanut calcium deficiency tolerance based on pod fullness, the process is as follows: Figure 1 As shown, the specific steps are as follows: S1. Plant the peanut germplasm resources or varieties to be tested under calcium deficiency stress conditions until maturity; wherein, calcium deficiency stress conditions refer to: natural field conditions in which the exchangeable calcium content in the soil is less than 400 mg / kg, or a controlled culture environment using nutrient solution with a calcium concentration of less than 2.0 mmol / L. S2. Harvest the pods of the peanut germplasm resources or varieties, and determine the kernel weight percentage (W1) and the kernel weight percentage (W2) of the pods. When calculating the pod-to-kernel weight ratio (W1) of the same peanut variety, the method for determining W1 is as follows: weigh 100 typical and uniform pods (M1, in g), peel them, and weigh the kernels (M2, in g); the formula for calculating W1 is as follows: Formula I; When calculating the kernel weight ratio (W1) of a single peanut plant, the method for determining W1 is as follows: select 20 typical and uniform pods and weigh them (M1, in g), peel them and weigh the kernels (M2, in g), and calculate according to Formula I; When calculating the percentage of full-shell kernels (W2) of the same peanut variety, the method for determining W2 is as follows: From the harvested pods, select full-shell pods that are plump, have clear reticulations, are free of disease spots, and are typical and uniform. Weigh 500g of each pod, and after shelling, weigh the kernels as N1. The unit is g. The formula for calculating W2 is as follows: Formula II; When calculating the percentage of full-shelled peanut kernels (W2) per peanut plant, the method for determining W2 is as follows: Weigh all full-shelled pods (selected from harvested pods that are plump, have clear reticulations, are free of disease spots, and are of typical uniformity) per plant (N2), select 20 typical uniform pods and weigh them (M1), shell them and weigh the kernels (M2), and shell the remaining full-shelled pods and weigh the kernels (N0); the formula for calculating W2 is shown below: Formula III; S3. Calculate the pod fullness decay index (SDI) of the peanut germplasm resource or variety. The SDI calculation formula is as follows: Formula IV; Wherein, SDI is the pod fullness decay index; W1 is the pod kernel weight percentage; W2 is the full pod kernel weight percentage. S4. Evaluate the calcium deficiency tolerance of the germplasm resource or variety based on the SDI value. The lower the SDI value, the stronger the calcium deficiency tolerance of the germplasm resource or variety.

[0031] Example 2: A method for screening calcium-deficient peanut varieties A method for screening calcium-deficient peanut varieties involves using the aforementioned method to calculate the SDI value of the peanut germplasm resource or variety to be tested, and comparing the SDI value with a preset threshold T. If SDI ≤ T, the variety is determined to be a calcium-deficient peanut variety. The preset threshold T ranges from 0.20 to 0.35. Preferably, the preset threshold T is 0.25.

[0032] Example 3: Evaluation of calcium deficiency tolerance of 32 peanut varieties using the method provided in Example 1 of this invention. 1. Experimental Conditions: The experiment was conducted in 2021 in red soil dryland of Liujiazhan Township, Yujiang District, Jiangxi Province. The soil exchangeable calcium content was 85 mg / kg, which is typical of calcium-deficient soil. A completely randomized block design was used, with each plot measuring 10m². 2 The experiment was repeated three times. The test materials consisted of 32 representative peanut varieties, which were the main varieties promoted in the Yangtze River Basin in recent years and the backbone parents in regional trials. These varieties were provided by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences. Conventional field management was implemented, and all nutrients except calcium were sufficient.

[0033] 2. Measurement and Calculation Methods: (1) Harvest representative plants from each plot at maturity and dry them to a safe moisture content.

[0034] (2) Determine the weight ratio of pod kernels W1: Weigh 100 typical and uniform pods (M1), peel them and weigh the kernels (M2). The formula for calculating W1 is the same as Formula I in Example 1.

[0035] (3) Determine the weight ratio of the kernel of the full pod W2: From the same material, select the pods that are full, have clear netting, and are undamaged and have a typical and consistent appearance as "full pods", weigh 500g, and weigh the kernel after shelling as N1. The formula for calculating W2 is the same as Formula II in Example 1.

[0036] (4) Calculate the pod fullness decay index (SDI), using the same formula as in Example 1, Formula IV.

[0037] 3. Results and Analysis: 3.1 The calculation results of W1, W2 and SDI are shown in Tables 1-2 and 2. Figure 2 As shown in Tables 1-2 and... Figure 2 It can be seen that the SDI values ​​of different varieties vary greatly, ranging from 0.164 (Ganhua 14-02) to 0.865 (Zhuhua 6). This indicates that under the same calcium-deficient environment, there are essential genetic differences in the degree of kernel abortion in "full-fruited" kernels of different varieties.

[0038] Table 1. Relevant indicators and SDI values ​​of some varieties of 32 peanut varieties under calcium deficiency conditions. Table 2. Relevant indicators and SDI values ​​of some varieties of 32 peanut varieties under calcium deficiency conditions. 3.2 Screening of calcium-deficiency tolerant varieties: The calcium deficiency tolerance threshold (T) was set at SDI ≤ 0.25. In this example, Ganhua 14-02 (0.164), 18-5105 (0.202), Luohua 106 (0.213), 153-57-2 (0.221), Wanhua 10 (0.226), 91-002 (0.236), and Wanhua 13 (0.237) were selected as calcium-deficiency tolerant varieties. Varieties with high SDI values, such as Zhuhua 6 (0.865), Qianhua 374 (0.696), and Ganhua 15-758 (0.621), were identified as calcium-sensitive varieties, and the kernel fullness of their kernels was severely reduced under calcium deficiency.

[0039] Example 4: Application of calcium deficiency tolerance screening method in F2 population of hybrid breeding In May 2022, a hybridization experiment was conducted using the calcium-deficiency-tolerant peanut variety Ganhua 14-02 (now registered as a non-major crop variety and named Ganhua 12) as the female parent and the high-yielding and highly resistant peanut variety Yuanza 9102 as the male parent. The resulting F1 seeds (18 seeds) were obtained by sowing in Nanchang, Jiangxi Province. In November 2022, these 18 F1 seeds were planted in Sanya, Hainan Province, and self-pollination yielded a segregating F2 population of 212 seeds. All experimental procedures involved conventional field management.

[0040] On May 9, 2023, 212 seeds from the previously obtained F2 population were planted in a red soil dryland plot (soil exchangeable calcium content of 102 mg / kg) at the Gao'an Base of the Jiangxi Academy of Agricultural Sciences. After maturity in 2023, individual plants were harvested, resulting in 196 individual plants (Tables 3-7). Conventional field management was used in the experiment. W1 and W2 were measured for each individual plant. (Due to the limited number of pods per plant, all full pods were weighed (N2), then 20 typical and uniform pods were weighed (M1), shelled, and the kernel weight was measured (M2). The remaining full pods were shelled and the kernel weight was measured (N0). W1 was calculated according to Formula I in Example 1, and W2 was calculated according to Formula III in Example 1. Then, the SDI value of each individual plant was calculated according to Formula IV in Example 1. The results are shown in Tables 3-7.)

[0041] The SDI values ​​of 196 individual plants showed a continuous distribution (Tables 3-7), with a range of 0.177-0.776, a mean of 0.356, and a standard deviation of 0.142. Based on the breeding objectives, 20 individual plants with the lowest SDI values ​​(i.e., SDI ≤ 0.223) were selected as superior calcium-deficiency tolerant plants. These superior plants not only had low SDI values, but their agronomic traits, such as yield per plant, were also examined simultaneously. The main stem height of these 20 individual plants ranged from 34 to 58 cm, the lateral branch length from 33 to 64 cm, the total number of branches from 6 to 8, the number of fruits from 31 to 53, and the yield per plant from 20 to 35 g (Table 8).

[0042] Table 3. Relevant indicators and SDI values ​​of some peanut plants under calcium deficiency conditions from 196 individual plants. Table 4. Relevant indicators and SDI values ​​of some peanut plants under calcium deficiency conditions from 196 individual plants. Table 5. Relevant indicators and SDI values ​​of some peanut plants under calcium deficiency conditions from 196 individual plants. Table 6. Relevant indicators and SDI values ​​of some peanut plants under calcium deficiency conditions from 196 individual plants. Table 7. Relevant indicators and SDI values ​​of some peanut plants under calcium deficiency conditions from 196 individual plants. Table 8. Yield and related indicators of 20 peanut plants under calcium deficiency conditions, and SDI value. Seeds from the superior plants listed in Tables 3-7 were harvested to form F3 lines. These F3 lines were then planted in Sanya, Hainan in November 2023 for self-pollination to form F4 lines. In May 2024, they were planted at the Gao'an base of the Jiangxi Academy of Sciences in Yichun for self-pollination to form F5 lines. Finally, in November 2024, they were planted in Sanya, Hainan for self-pollination to form F6 lines. All experimental procedures involved conventional field management.

[0043] Example 5: Production Validation of the Screened Strain In May 2025, field plot trials were conducted at the Gao'an base of the Yichun Institute of Technology in Jiangxi Province. Four stable low-calcium tolerant varieties with good yield traits, derived from the F6 line, were planted. The calcium-sensitive variety Ganhua 18 was used as a control. The trials included no calcium application (soil exchangeable calcium content of 98 mg / kg) and normal calcium application (50 kg / 667 m²). 2 Two calcium treatments were applied. Each plot was 10m². 2 The experiment was a randomized block design with three biological replicates, a total of 10 treatments, and 30 plots. The experimental protocol is shown in Table 9.

[0044] Table 9 Test Implementation Plan The results are shown in Table 10. The results show that, regardless of whether calcium was applied without treatment or under normal calcium application, the SDI values ​​of the four identified lines remained stable below 0.25. Under the no-calcium treatment, the four lines showed a yield increase of 59.91%-76.05% compared to the control (Ganhua 18), demonstrating a highly significant effect. However, under normal calcium application conditions, the yield increase was only 5.73%-11.86%. This fully demonstrates that the low-SDI varieties screened by the method of this invention have strong stable production capacity and application value in calcium-deficient soils.

[0045] Table 10. Peanut yield and related indicators and SDI value under different treatments The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for evaluating peanut calcium deficiency tolerance based on pod fullness, characterized in that, The method includes the following steps: Peanuts to be tested were grown under calcium deficiency stress until maturity; Harvest the peanut pods to be tested, and determine the weight percentage of kernels in the pods and the weight percentage of kernels in the full pods, respectively; The pod fullness decay index of the peanut to be tested is calculated based on the weight percentage of the pod kernels and the weight percentage of the full kernels. The calcium deficiency tolerance of the tested peanut was evaluated based on the pod fullness decay index.

2. The method according to claim 1, characterized in that, The lower the pod fullness decay index of the peanut being tested, the stronger its tolerance to calcium deficiency.

3. The method according to claim 1, characterized in that, The calcium deficiency stress conditions include natural field conditions where the exchangeable calcium content in the soil is less than 400 mg / kg, or a controlled culture environment using a nutrient solution with a calcium concentration of less than 2.0 mmol / L.

4. The method according to claim 1, characterized in that, The formula for calculating the weight percentage of the pod kernels is as follows: ； Wherein, W1 is the weight percentage of pod kernels; M1 is the weight of pods in g; and M2 is the weight of shelled kernels in g.

5. The method according to claim 1, characterized in that, The formula for calculating the weight percentage of the plump kernels is as follows: ； Where W2 is the weight percentage of full-bodied kernels; N1 is the weight of shelled kernels, in g; Alternatively, the formula for calculating the weight percentage of the full-bodied kernels is as follows: ； Wherein, W2 is the weight percentage of full pods; N0 is the weight of shelled kernels of full pods remaining after removing 20 pods per plant, in g; M2 is the weight of shelled kernels of 20 pods per plant, in g; and N2 is the weight of all full pods per plant, in g.

6. The method according to claim 1, characterized in that, The formula for calculating the pod fullness decay index is as follows: ； Wherein, SDI is the pod fullness decay index; W1 is the pod kernel weight percentage; and W2 is the full pod kernel weight percentage.

7. The application of the method according to any one of claims 1-6 in screening calcium-deficient peanut varieties.

8. A method for screening calcium-deficient peanut varieties, characterized in that, The method includes calculating the pod fullness decay index of the peanut to be tested using the method described in any one of claims 1-6, and comparing the obtained pod fullness decay index with a preset threshold T; If the pod fullness decay index is less than or equal to the preset threshold T, the peanut to be tested is determined to be a calcium-deficient peanut variety; the preset threshold T ranges from 0.20 to 0.

35.

9. The application of the method according to any one of claims 1-6 in flowering breeding, characterized in that, The flowering breeding includes early screening of calcium-deficiency-tolerant individual plants or lines from hybrid offspring populations, induced mutation populations, or naturally mutated populations.

10. A method for breeding calcium-deficient peanut varieties, characterized in that, Includes the following steps: Hybridization was carried out using calcium-tolerant peanut germplasm as parents, or breeding populations were created using mutagenesis techniques; the breeding populations were then planted under calcium-deficiency stress conditions. Using the method described in any one of claims 1-6, plants with low pod fullness decay index are screened out; The selected plants were propagated over multiple generations and their trait stability was assessed, ultimately resulting in a new peanut variety tolerant to calcium deficiency.

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