Method for evaluating salt and alkali tolerance of different leymus chinensis germplasm resources in field

Abstract

The application belongs to the field of crop genetic breeding, and particularly relates to a method for evaluating the salt and alkali tolerance of different sheep grass germplasm resources in a field. The application is to solve the problem of large trait data error caused by uneven initial sheep grass tillering seedlings and uneven transplanting damage in the existing field evaluation. The method is as follows: one, first uniform treatment; two, first transplanting; three, second uniform treatment; four, second transplanting; five, salt and alkali tolerance phenotype observation; and six, salt and alkali tolerance evaluation of salt and alkali tolerance plant germplasm. The core of the application is to perform two times of uniform treatment and two times of transplanting on the sheep grass tillering seedlings, so that the starting uniformity of the test plants is greatly improved, and the systematic error caused by the transplanting link is effectively controlled.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and specifically relates to a method for field evaluation of the salt and alkali tolerance of different Leymus chinensis germplasm resources. Background Technology

[0002] Leymus chinensis, due to its superior quality and strong resilience, has become a pioneer forage species for improving soda-alkali soils in Northeast China. However, the lack of field assessment techniques for salt tolerance severely restricts its efficient breeding and application. As a self-incompatible perennial rhizomatous forage, Leymus chinensis suffers from "three lows"—low germination rate, low heading rate, and low seed setting rate—for field assessment must rely on asexual reproduction of tillers. Furthermore, its underground rhizomes grow horizontally, and new branches can sprout from the nodes, leading to variations in rhizome condition and length among transplanted seedlings, directly affecting the phenotypic consistency of key traits such as tiller number and plant height. Therefore, ensuring the initial uniformity of transplanted tillers is the primary prerequisite and fundamental guarantee for accurate assessment. Summary of the Invention

[0003] The purpose of this invention is to solve the problem of large errors in trait data caused by uneven initial tillering and transplanting damage in existing field assessments, and to provide a method for field assessment of the salt and alkali tolerance of Leymus chinensis germplasm resources.

[0004] The following methods were used for field assessment of the salt tolerance of different Leymus chinensis germplasm resources.

[0005] I. First homogenization treatment: Leymus chinensis has a well-developed root system and can reproduce asexually through underground rhizomes. After the Leymus chinensis turns green again, select 4-5 tillers with uniform growth, requiring a root length of 20 cm ± 5 cm, to complete the first homogenization treatment of Leymus chinensis germplasm. The first homogenization treatment is to reduce the initial value error between different Leymus chinensis germplasm. Selecting a uniform number of tillers with similar root lengths is to ensure that new tillers grow in a more uniform manner after transplanting, for subsequent salt and alkali tolerance assessment.

[0006] II. First Transplanting: The Leymus chinensis germplasm treated in the first homogenization was transplanted into normal soil and allowed to grow for 45±1 days, completing the first transplanting. At this time, all transplanted tillers had survived and produced multiple new tillers. Their roots were relatively loosely intertwined, making it easier to transplant the new tillers from the soil and reducing root damage. Furthermore, the newly grown tillers ensured greater consistency in the initial state of the Leymus chinensis. (The first homogenization treatment and transplanting ensured the growth of new tillers in a consistent state, which would be used for subsequent transplanting and salt tolerance assessment.)

[0007] III. Second homogenization treatment: After 45±1 days of growth, the newly transplanted Leymus chinensis tillers will produce new tillers. Select new tillers with consistent growth, and ensure that the root length is 6 cm±1 cm and the plant height is 10 cm±1 cm. (The second homogenization treatment is to further reduce the initial error in the growth status of the newly grown tillers.)

[0008] IV. Second Transplanting: The newly transplanted Leymus chinensis germplasm tillers, after undergoing two homogenization treatments, were transplanted into normal soil and saline-alkali soil respectively, completing the second transplanting. These newly transplanted tillers can then be used for subsequent assessment of salt tolerance.

[0009] V. Observation of salt and alkali tolerance phenotype: After the second transplanting, the Leymus chinensis tillers continued to grow in normal soil and saline-alkali soil for 60±1 days. It was clearly observed that the plant height, number of tillers, number of leaves, leaf length and leaf width of the plants in saline-alkali soil were significantly lower than those of the plants in normal soil, and were accompanied by yellowing of leaves.

[0010] VI. Evaluation of salt and alkali tolerance of Leymus chinensis germplasm: The plant height, number of tillers, number of leaves, leaf length, leaf width and chlorophyll content of each Leymus chinensis germplasm were statistically analyzed under normal soil and saline-alkali soil conditions. The ratio of each trait value under saline-alkali conditions to the corresponding value under normal conditions was used as the evaluation index of salt and alkali tolerance. The ratio ranged from 0 to 1. The closer to 1, the stronger the salt and alkali tolerance, and the closer to 0, the weaker the salt and alkali tolerance.

[0011] The pH value of the saline-alkali soil mentioned in step four is 9.0~9.2. Under this pH condition, the survival of Leymus chinensis can be guaranteed, and the salinity of Leymus chinensis germplasm can also be identified.

[0012] Step 6: Use a SPAD 502 Plus chlorophyll content analyzer to determine the chlorophyll content.

[0013] This invention controls errors through a systematic process of "two homogenization treatments" and "two transplantings." First, the Leymus chinensis tillers undergo a first homogenization treatment and are transplanted to a prepared substrate. After 45 days of recovery, tillers with consistent growth are selected for a second transplanting, planting them in both saline-alkali soil (pH = 9.0~9.2) and normal soil to ensure initial consistency. Subsequently, the number of tillers, plant height, number of leaves, leaf length and width, and chlorophyll content are statistically analyzed under both growing conditions. Finally, the salt tolerance of the Leymus chinensis germplasm is comprehensively evaluated by calculating the ratio of each trait in saline-alkali soil to that in normal soil.

[0014] The core of this invention lies in performing two "homogenization treatments" and "two transplants" on Leymus chinensis tiller seedlings. This process greatly improves the initial uniformity of the experimental plants and effectively controls the systematic errors caused by the transplanting process, thus providing technical support for the accurate and reliable identification of the salt and alkali tolerance of Leymus chinensis germplasm resources.

[0015] The beneficial effects of this invention are:

[0016] (1) Addressing industry pain points: Soil salinization is a major global abiotic stress. Leymus chinensis exhibits strong salt and alkali tolerance, but research on genes regulating salt and alkali tolerance is scarce. GWAS (genome-wide association study) is one of the main strategies for discovering genes regulating salt and alkali tolerance in Leymus chinensis. However, due to its self-incompatibility, field assessment of Leymus chinensis heavily relies on asexual reproduction of tillers, and the initial uniformity of transplanted seedlings is difficult to control, becoming a bottleneck restricting the application of efficient gene discovery strategies in GWAS. This invention directly addresses this core challenge and provides a reliable solution.

[0017] (2) This method is innovative and reliable: The "two transplants and two homogenization treatments" method pioneered by this invention systematically eliminates the initial differences caused by the growth characteristics of the underground rhizomes of sheep grass, and fundamentally ensures the uniformity of the experimental materials.

[0018] (3) Enhancing data value: By ensuring the uniformity of transplanted seedlings, this invention significantly improves the reliability and accuracy of key phenotypic data such as the number of tillers, number of leaves, leaf length, leaf width and chlorophyll content of Leymus chinensis obtained in the field, thereby providing a solid technical guarantee for high-quality GWAS analysis, accurate mining of salt-tolerant genes and cultivation of superior germplasm resources. Attached Figure Description

[0019] Figure 1 This is a phenotypic diagram of the aboveground parts of Leymus chinensis after it turned green again in Experiment 1;

[0020] Figure 2 This is a diagram showing the overall plant morphology (above-ground and below-ground parts) of Leymus chinensis after it turned green again in Experiment 1.

[0021] Figure 3 This is a close-up of the underground rhizome structure of sheep grass in Experiment 1;

[0022] Figure 4 This is the initial state of the Leymus chinensis tillers after the first homogenization treatment in Experiment 1;

[0023] Figure 5 This is the growth status of Leymus chinensis 50 days after its first transplanting in Experiment 1;

[0024] Figure 6 This is the state of Leymus chinensis after the second homogenization treatment in Experiment 1;

[0025] Figure 7This is the growth status of Leymus chinensis one day after the second transplanting in Experiment 1;

[0026] Figure 8 This is the state of Leymus chinensis after 60 days of growth in normal and saline-alkali soils in Experiment 1 (normal soil on the left, saline-alkali soil on the right).

[0027] Figure 9 This is a comparison of leaf color phenotypes of Leymus chinensis after 60 days of growth in normal and saline-alkali soils in Experiment 1. Detailed Implementation

[0028] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0029] Specific Implementation Method 1: The method used in this implementation method for field evaluation of the salt tolerance of different Leymus chinensis germplasm resources is as follows:

[0030] I. First homogenization treatment: After the Leymus chinensis turns green again, select 4-5 tillers with uniform growth and root length of 20cm±5cm to complete the first homogenization treatment of Leymus chinensis germplasm.

[0031] II. First transplanting: The sheepgrass germplasm that had undergone the first homogenization treatment was transplanted into normal soil and allowed to grow for 45±1 days to complete the first transplanting.

[0032] 3. Second homogenization treatment: After the first transplanting, select newly grown tillers with consistent growth status, and the tillers should have a root length of 6 cm ± 1 cm and a plant height of 10 cm ± 1 cm to complete the second homogenization treatment.

[0033] IV. Second transplanting: The new tillers of Leymus chinensis germplasm that have undergone two homogenization treatments are transplanted into normal soil and saline-alkali soil respectively to complete the second transplanting.

[0034] V. Observation of salt and alkali tolerance phenotype: After the second transplanting, the tillering seedlings of Leymus chinensis continued to grow for 60±1 days in normal soil and saline-alkali soil;

[0035] VI. Evaluation of salt and alkali tolerance of Leymus chinensis germplasm: The plant height, number of tillers, number of leaves, leaf length, leaf width and chlorophyll content of Leymus chinensis germplasm under normal soil and saline-alkali soil conditions were counted respectively. The ratio of each trait value under saline-alkali conditions to the corresponding value under normal conditions was used as the evaluation index of salt and alkali tolerance. The ratio ranged from 0 to 1. The closer to 1, the stronger the salt and alkali tolerance, and the closer to 0, the weaker the salt and alkali tolerance.

[0036] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the pH value of the saline-alkali soil described in step four is 9.0~9.2. Everything else is the same as in Specific Implementation Method One.

[0037] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that step six uses a SPAD 502 Plus chlorophyll content meter to measure the chlorophyll content. Everything else is the same as in Specific Implementation Method One or Two.

[0038] The following experiments were used to verify the effectiveness of the invention:

[0039] Experiment 1:

[0040] The following methods were used for field assessment of the salt tolerance of different Leymus chinensis germplasm resources.

[0041] I. First homogenization treatment: After the Leymus chinensis turns green again, select 4-5 uniformly growing tillers with a root length of 25cm to complete the first homogenization treatment of Leymus chinensis germplasm (see...). Figure 1-4 );

[0042] II. First Transplanting: The homogenized Leymus chinensis germplasm was transplanted into normal soil and allowed to grow for 45 days to complete the first transplanting (see...). Figure 5 );

[0043] III. Second homogenization treatment: After the first transplanting, select tillers with uniform growth, root length of 6cm, and plant height of 10cm, and complete the second homogenization treatment (see...). Figure 6 );

[0044] IV. Second Transplanting: The sheepgrass seedlings that have undergone two homogenization treatments are transplanted into normal soil and saline-alkali soil respectively to complete the second transplanting (see...). Figure 7 );

[0045] V. Observation of Salt and Alkali Tolerance Phenotypic: After the second transplanting, the Leymus chinensis tillers continued to grow in normal soil and saline-alkali soil for 60±1 days. It was clearly observed that the plant height, number of tillers, number of leaves, leaf length, and leaf width of the plants in saline-alkali soil were significantly lower than those in normal soil, accompanied by leaf yellowing (see...). Figure 8 , Figure 9 );

[0046] VI. Evaluation of salt and alkali tolerance of Leymus chinensis germplasm: The plant height, number of tillers, number of leaves, leaf length, leaf width and chlorophyll content of each Leymus chinensis germplasm were statistically analyzed under normal soil and saline-alkali soil conditions. The ratio of each trait value under saline-alkali conditions to the corresponding value under normal conditions was used as the evaluation index of salt and alkali tolerance. The ratio ranged from 0 to 1. The closer to 1, the stronger the salt and alkali tolerance, and the closer to 0, the weaker the salt and alkali tolerance.

[0047] The pH value of the saline-alkali soil described in step four is 9.0.

[0048] Step 6: Use a SPAD 502 Plus chlorophyll content analyzer to determine the chlorophyll content.

Claims

1. A method for field evaluation of the salt and alkali tolerance of different Leymus chinensis germplasm resources, characterized in that... The method for field evaluation of the salt tolerance of different Leymus chinensis germplasm resources is as follows: I. First homogenization treatment: After the Leymus chinensis turns green again, select 4-5 tillers with uniform growth and root length of 20 cm ± 5 cm to complete the first homogenization treatment of Leymus chinensis germplasm. II. First transplanting: The sheepgrass germplasm that had undergone the first homogenization treatment was transplanted into normal soil and allowed to grow for 45±1 days to complete the first transplanting.

3. Second homogenization treatment: After the first transplanting, select newly grown tillers with consistent growth status, and the tillers should have a root length of 6 cm ± 1 cm and a plant height of 10 cm ± 1 cm to complete the second homogenization treatment. IV. Second transplanting: The new tillers of Leymus chinensis germplasm that have undergone two homogenization treatments are transplanted into normal soil and saline-alkali soil respectively to complete the second transplanting. V. Observation of salt and alkali tolerance phenotype: After the second transplanting, the tillering seedlings of Leymus chinensis continued to grow for 60±1 days in normal soil and saline-alkali soil; VI. Evaluation of salt and alkali tolerance of Leymus chinensis germplasm: The plant height, number of tillers, number of leaves, leaf length, leaf width and chlorophyll content of Leymus chinensis germplasm under normal soil and saline-alkali soil conditions were counted respectively. The ratio of each trait value under saline-alkali conditions to the corresponding value under normal conditions was used as the evaluation index of salt and alkali tolerance. The ratio ranged from 0 to 1. The closer to 1, the stronger the salt and alkali tolerance, and the closer to 0, the weaker the salt and alkali tolerance. The pH value of the saline-alkali soil described in step four is 9.0~9.

2.

2. The method for field evaluation of salt and alkali tolerance of different Leymus chinensis germplasm resources according to claim 1, characterized in that... Step 6: Use a SPAD 502 Plus chlorophyll content analyzer to determine the chlorophyll content.

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