Method for identifying low cadmium accumulation characteristic of rice sterile line and application of method

Abstract

The invention belongs to the technical field of crop heavy metal accumulation phenotype identification and crop breeding, and particularly relates to a method for identifying the low cadmium accumulation characteristic of a rice sterile line and application of the method. According to the method disclosed by the invention, simplification, standardization and high efficiency of cadmium low accumulation characteristic phenotype identification of the sterile line are realized through a standardized identification environment and diversified detection samples which are easy to obtain, such as a small amount of self-fertilized or outfertilized rice and unfertilized empty grains or rice ears or leaves; important support is provided for cultivation and official certification of the low-cadmium sterile line, so that breeding and large-area popularization and application of low-cadmium hybrid rice varieties are improved.

Description

Technical Field

[0001] This invention belongs to the field of crop heavy metal accumulation phenotypic identification and crop breeding technology, specifically involving a method for identifying the low cadmium accumulation characteristics of rice male-sterile lines and its application. Background Technology

[0002] Hybrid rice varieties possess significant advantages such as high yield and stress resistance, making them crucial for increasing rice yield per unit area and total output in my country. The breeding of hybrid rice varieties is inseparable from the development of sterile lines. Low-cadmium rice is currently the most economical and effective way to address cadmium pollution in rice, and the breeding of low-cadmium hybrid rice varieties is a key measure to achieve increased yields. The breeding of low-cadmium hybrid rice varieties requires that both the male and female parents (sterile lines) possess low cadmium accumulation characteristics; therefore, the cultivation and identification of low-cadmium sterile lines have become an indispensable part of low-cadmium hybrid rice breeding.

[0003] Currently, the technology for identifying the low cadmium accumulation characteristics of low cadmium rice varieties is well-developed, and the phenotype of low cadmium accumulation characteristics in paddy rice or brown rice is usually used to characterize the variety's traits. However, due to reasons such as the inability of male-sterile lines to naturally self-pollinate or excessively low seed setting rates, making it difficult to obtain sufficient paddy rice samples, or due to the stringent conditions and complex procedures for restoring fertility in male-sterile lines, the identification of the phenotype of low cadmium accumulation characteristics in low-cadmium male-sterile lines is quite challenging, and in most cases, it remains at the level of indirect genotyping. However, since genotyping cannot directly and fully reflect the characteristics of the variety itself, it cannot be used as a direct basis for official identification of male-sterile lines. On the other hand, existing methods for restoring the fertility of male-sterile lines and evaluating the phenotype of low cadmium accumulation characteristics in field environments through complex measures are easily affected by natural rainfall, soil moisture and cadmium content, and pH uniformity, resulting in poor annual and regional repeatability of identification results, low identification efficiency, and weak support for the breeding and identification of male-sterile lines. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for identifying the low cadmium accumulation characteristics of rice male-sterile lines and its application. The method, through a standardized identification environment and diverse and easily obtainable test samples, achieves simplified, standardized, and highly efficient identification of the cadmium accumulation phenotype of rice male-sterile lines.

[0005] This invention provides a method for identifying the low cadmium accumulation characteristics of rice male-sterile lines, comprising the following steps: 1) The rice sterile line to be identified was raised to obtain rice seedlings; 2) Transplant the rice seedlings into a rain-sheltered and light-permeable identification pond, and then carry out field management until maturity; the identification pond has smooth watering and drainage. The soil in the identification pool is natural soil excavated from the topsoil of paddy fields. The total cadmium content of the soil in the identification pool is 1.0~2.0 mg / kg or 1.0~1.7 mg / kg, and the thickness of the soil in the identification pool is 0.15~0.4m. The field management includes irrigation management; The irrigation management includes: maintaining a water level of 1-3 cm in the identification pond after the rice seedlings are transplanted and before the field is dried at the end of the tillering stage; drying the field for 7-9 days at the end of the tillering stage; and cyclical management of irrigation, drainage, drying, re-irrigation, and then drainage and drying the field after the field is dried at the end of the tillering stage until the rice is harvested; maintaining a water level of 2-3 cm in the identification pond for 6-24 hours each time the field is irrigated, and drying the field until it is no longer muddy when stepped on; 3) Sampling and testing of cadmium content at maturity. When the cadmium content of the rice sample is ≤0.2mg / kg, the rice male-sterile line to be identified is determined to have low cadmium accumulation characteristics; otherwise, the rice male-sterile line to be identified does not have low cadmium accumulation characteristics. The rice samples include one or more of the following: self-pollinated full grains, cross-pollinated full grains, unfertilized empty grains, panicles, and leaves.

[0006] Preferably, the leaves in the rice sample described in step 3) include leaves from any location other than axillary bud regeneration seedlings, or a mixture of leaves from multiple locations.

[0007] Preferably, the rain-sheltered and light-transmitting identification pool is equipped with a sun panel with rain-proof function; the rain-sheltered pool is equipped with water filling and / or drainage facilities.

[0008] Preferably, the width of the identification pool is 1.5~3.0m and the length is 5~50m; the long side wall of the identification pool is provided with two rows of seepage holes, the height of the lower row of seepage holes is level with the bottom of the identification pool, the height of the upper row of seepage holes is level with the top surface of the soil in the identification pool, the distance between adjacent seepage holes in the same row is 0.15~0.3m, and the diameter of each seepage hole is 2~3cm; The identification pool has a row of water inlet holes on its long side wall. The height of the water inlet holes is 0-20cm higher than the upper row of seepage holes. The distance between adjacent water inlet holes in the same row is 0.15-0.3m, and the diameter of each water inlet hole is 2-3cm.

[0009] Preferably, the cadmium content detection includes the following steps: drying the rice sample and then pulverizing it to obtain a rice powder sample; The cadmium content in the rice powder sample was determined by graphite furnace atomic absorption spectrophotometry.

[0010] Preferably, during the sampling and cadmium content detection, no fewer than three biological replicates are set up for the same rice sample, and the replicate with the highest cadmium content is used to determine the low cadmium accumulation characteristic.

[0011] Preferably, the seedling raising method described in step 1) includes tray seedling throwing and / or water seedling raising.

[0012] Preferably, the method includes simultaneously identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines; when simultaneously identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines, each rice male-sterile line to be identified is planted with no less than three biological replicates, each biological replicate has no less than 10 holes, and one rice seedling is planted in each hole.

[0013] Preferably, the method further includes planting a non-cadmium-low rice sterile line as a control.

[0014] The present invention also provides the application of the method described in the above technical solution in the breeding of low-cadmium rice varieties.

[0015] Beneficial effects: This invention provides a method for identifying the low cadmium accumulation characteristic of rice male-sterile lines and its application. The method, through a standardized identification environment and diverse and easily obtainable test samples, simplifies, standardizes, and highly efficiencies the identification of the low cadmium accumulation phenotype in male-sterile lines. Specific advantages are as follows: 1) While existing methods for identifying the low cadmium accumulation characteristics of rice male-sterile lines using genotyping offer advantages such as speed, efficiency, relatively low cost, and minimal environmental impact, their results can only indirectly infer the genetic potential of known genes for low cadmium accumulation in the tested materials. This invention, however, utilizes phenotypic identification to evaluate the low cadmium accumulation characteristics of rice male-sterile lines. By planting the tested materials in a precisely controlled environment until maturity and then measuring the cadmium content in the rice grains or leaves, it directly demonstrates the low cadmium accumulation characteristics of rice male-sterile lines under actual planting conditions, reflecting the final result of the interaction between all genes (known and unknown) of the tested materials and the environment. Therefore, compared to genotyping, phenotypic identification results are more accurate and reliable, serving as the sole legal basis for variety approval and food safety evaluation.

[0016] 2) Existing methods for evaluating the phenotypic characteristics of low cadmium accumulation in rice male-sterile lines in field environments are susceptible to the effects of natural rainfall, soil moisture, cadmium content, and pH value uniformity, resulting in poor repeatability of identification results across years and regions and low identification efficiency. This invention achieves accurate identification under controlled conditions by using standardized rain-sheltered and light-permeable cadmium-conducting paddy fields (rain-sheltered cadmium identification ponds), resulting in better reliability and repeatability of identification results and higher identification efficiency.

[0017] 3) Existing methods for determining whether a test material has low cadmium accumulation characteristics by detecting the cadmium content in the brown rice of sterile lines require planting restorer lines simultaneously with the sterile lines to enable the three-line sterile lines to produce grains, or artificially controlling environmental conditions at specific developmental stages to restore fertility and produce grains in light- and temperature-sensitive sterile lines. These methods place high demands on the identification platform (requiring environmental control equipment for light or temperature), are complex to operate, technically challenging, and costly. Furthermore, the identification of low cadmium accumulation characteristics in three-line and two-line sterile lines requires different environmental and technical solutions, making widespread application difficult. This invention determines the low cadmium accumulation characteristics of test materials by directly detecting the cadmium content in the rice grains, empty grains, panicles, or leaves of sterile lines (both three-line and two-line sterile lines) 35-45 days after heading. This avoids the complex operation of restoring fertility in sterile lines, has lower facility requirements and costs, and is technically simpler and easier to implement. Detailed Implementation

[0018] This invention provides a method for identifying the low cadmium accumulation characteristics of rice male-sterile lines, comprising the following steps: 1) The rice sterile line to be identified was raised to obtain rice seedlings; 2) Transplant the rice seedlings into a rain-sheltered and light-permeable identification pond, and then carry out field management until maturity; the identification pond has smooth watering and drainage. The soil in the identification pool is natural soil excavated from the topsoil of paddy fields. The total cadmium content of the soil in the identification pool is 1.0~2.0 mg / kg or 1.0~1.7 mg / kg, and the thickness of the soil in the identification pool is 0.15~0.4m. The field management includes irrigation management; The irrigation management includes: maintaining a water level of 1-3 cm in the identification pond after the rice seedlings are transplanted and before the field is dried at the end of the tillering stage; drying the field for 7-9 days at the end of the tillering stage; and cyclical management of irrigation, drainage, drying, re-irrigation, and then drainage and drying the field after the field is dried at the end of the tillering stage until the rice is harvested; maintaining a water level of 2-3 cm in the identification pond for 6-24 hours each time the field is irrigated, and drying the field until it is no longer muddy when stepped on; 3) Sampling and cadmium content detection at maturity: when the cadmium content of the rice sample is ≤0.2mg / kg, the rice male-sterile line to be identified is determined to have low cadmium accumulation characteristics; otherwise, the rice male-sterile line to be identified does not have low cadmium accumulation characteristics. The rice samples include one or more of the following: self-pollinated full grains, cross-pollinated full grains, unfertilized empty grains, panicles, and leaves.

[0019] As one implementation method, the identification pool of the present invention is provided with a sun panel with rain protection function above it; the identification pool is equipped with water filling and / or drainage facilities; specifically, in the embodiments of the present invention, the method uses an identification pool for identification, and a rain-proof sunshade is built above the identification pool, and water filling and drainage are convenient and quick.

[0020] Simultaneously or subsequently, the present invention cultivates rice seedlings of the rice male-sterile lines to be identified at the selected implementation location. As one implementation method, the seedling cultivation can be tray transplanting and / or water-based seedling cultivation, with tray transplanting being a more specific method. As another implementation method, seedling cultivation can be performed simultaneously on multiple rice male-sterile line varieties to be identified, with each variety sown in one tray, each tray containing at least 50 consecutive holes, and one germinated rice seed sown per hole. As one implementation method, the present invention selects robust seedlings for manual transplanting with soil attached; as another implementation method, before transplanting, the planting rows are planned using a row marker or similar device. In the present invention, the strict limitation of the seedling cultivation parameters can prevent seedling mixing and thus avoid errors in the identification results.

[0021] After seedling cultivation, the rice seedlings are transplanted into an identification pond, and then field management is carried out until maturity. As one embodiment, the transplanting specifications are not particularly demanding; for example, a spacing of 20cm x 20cm can be used. As another embodiment, the identification pond has a width of 1.5-3.0m and a length of 5-50m. Two rows of drainage holes are provided on the long side wall of the identification pond. The lower row of drainage holes is level with the bottom of the identification pond, and the upper row is level with the surface of the soil in the identification pond. The distance between adjacent drainage holes in the same row is 0.15-0.3m, and the diameter of each drainage hole is 2-3cm. A row of water inlets is provided on the long side wall of the identification pond. The height of the water inlets is 0-20cm higher than the upper row of drainage holes. The distance between adjacent water inlets in the same row is 0.15-0.3m, and the diameter of each water inlet is 2-3cm. In this invention, as one embodiment, the width of the identification pool is 2.0~2.5m, and the length is 10~40m, more specifically 20~30m. As one embodiment, the distance between adjacent seepage holes in the same row is 0.2~0.25m, and the diameter of each seepage hole is 2.5~3cm. As one embodiment, the distance between adjacent inlet holes in the same row is 0.2~0.25m, and the diameter of each inlet hole is 2.5~3cm. In this invention, this elongated structure facilitates rapid irrigation and drainage in the central area of ​​the identification pool, helps ensure uniform soil moisture content in all parts of the identification pool, and improves the accuracy and repeatability of the identification results. In this invention, the total cadmium content in the soil of the identification pool is 1.0~2.0 mg / kg or 1.0~1.7 mg / kg, more preferably 1.5~1.7 mg / kg. The 1.0~2.0 mg / kg range is suitable for identifying the low cadmium accumulation characteristics of rice male-sterile lines using self-pollinated full-grain rice, cross-pollinated full-grain rice, unfertilized empty grains, or rice panicles as test samples. The 1.0~1.7 mg / kg range is suitable for identifying the low cadmium accumulation characteristics of rice male-sterile lines using leaves as test samples. When the total cadmium content in the soil of the identification pool is below 1 mg / kg or above 2 mg / kg, the accuracy of the identification results will decrease. In this invention, the soil thickness of the identification pool is 0.15~0.4 m. A thickness less than 0.15 m is not conducive to the healthy growth of rice and cannot guarantee the accuracy of the identification results. In one implementation method, the pH value of the soil in the identification pool is 5.0-6.5; this pH value is suitable for rice growth and is also the common pH value of natural paddy field soil, making the method of the present invention closer to the field production environment. In the present invention, the cadmium content, pH value, and moisture content of the soil in the identification pool are uniform, which helps to ensure the repeatability of the identification results.

[0022] In one implementation, the method includes simultaneously identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines. When simultaneously identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines, each rice male-sterile line to be identified is planted with no fewer than three biological replicates, each biological replicate with no fewer than 10 holes, and one rice seedling is planted in each hole. In another implementation, when the multiple rice male-sterile lines include early, mid, and late rice male-sterile lines, the three types of male-sterile lines are planted in separate areas and groups, with one non-low cadmium variety planted in each group as a control.

[0023] In this invention, field management includes irrigation management. The irrigation management includes: maintaining a water level of 1-3 cm in the identification pond after rice seedling transplanting until the end of tillering and before drying the field; drying the field for 7-9 days at the end of tillering; and cyclical management of irrigation, drainage, drying, re-irrigation, and then drainage and drying the field after drying the field at the end of tillering until rice harvest; maintaining a water level of 2-3 cm in the identification pond for 6-24 hours each time during irrigation, and drying the field until it is no longer muddy underfoot each time. As one embodiment, maintaining a water level of 2-3 cm in the identification pond for 6-12 hours each time during irrigation. As one embodiment, the irrigation water source is a clean, pollution-free water source. In this invention, the irrigation management settings ensure uniformity and standardization of soil moisture status, while ensuring that the cadmium accumulation characteristics of the identification materials are fully reflected, ensuring the accuracy and repeatability of the identification results.

[0024] In one implementation method, the field management in this invention also includes fertilization management and pest and weed control. The steps of fertilization management and pest and weed control are not particularly required and can be carried out in accordance with conventional field production methods.

[0025] After the rice seedlings have developed to maturity, the present invention takes rice samples at maturity to detect cadmium content. When the cadmium content of the rice sample is ≤0.2mg / kg, it is determined that the rice sterile line to be identified has low cadmium accumulation characteristics; otherwise, the rice sterile line to be identified does not have low cadmium accumulation characteristics.

[0026] In one implementation method, the sampling time in this invention is at the maturity stage, i.e., 35-45 days after the beginning of heading. In this invention, the rice sample includes one or more of the following: self-pollinated full-grain rice, outcrossed full-grain rice, unfilled empty grains, panicles, and leaves. Further, the rice is selected in the order of self-pollinated full-grain rice, outcrossed full-grain rice, unfilled empty grains, panicles, and leaves, from front to back, to better characterize the cadmium accumulation characteristics of rice. Specifically, the cadmium content of full-grain rice is more representative than that of empty grains, which is more representative than that of panicles and leaves. Even if samples from earlier positions cannot be obtained normally, the test results of samples from later positions can indirectly reflect the low cadmium accumulation characteristics of the sterile line. In another implementation method, the leaves in the rice sample of this invention include leaves from any position other than axillary bud regeneration seedlings, or a mixture of leaves from multiple positions. This is because axillary bud regeneration seedlings are late-stage growth seedlings, not synchronized with the rice growth period, and cannot accurately represent the cadmium accumulation characteristics of rice. In one embodiment, the leaves include sword leaves; the growth time and location of sword leaves are closest to those of rice ears and rice grains, and their cadmium accumulation content is also likely to be closer to that of rice ears and rice grains; moreover, sword leaves are easier to sample than other leaves.

[0027] In one embodiment, the cadmium content detection of the present invention includes the following steps: drying and pulverizing the rice sample to obtain a rice powder sample; and detecting the cadmium content in the rice powder sample using graphite furnace atomic absorption spectrophotometry. In one embodiment, the drying method includes constant temperature forced-air drying at 80°C for 48-72 hours, more preferably 60-65 hours. In one embodiment, the particle size of the rice powder sample is 60-100 mesh. In one embodiment, the graphite furnace atomic absorption spectrophotometry method is performed according to the graphite furnace atomic absorption spectrophotometry method specified in GB 5009.15 to digest and detect the cadmium content in the sample. In one embodiment, during the cadmium content detection, at least three biological replicates are set up for the same rice sample, and the replicate with the highest cadmium content is used to determine the low cadmium accumulation characteristics; more preferably, three biological replicates are used.

[0028] The present invention also provides the application of the method described in the above technical solution in the breeding of low-cadmium rice varieties.

[0029] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Identification of the low-cadmium two-line sterile line Lian 1S: Experimental location: Phenotypic identification platform for low cadmium accumulation characteristics in rice, Hunan Academy of Agricultural Sciences, Chunhua Town, Changsha County.

[0031] Test materials: Longzhen 36S (non-low cadmium two-line sterile line), Lian 1S (low cadmium version of Longzhen 36S), Zhenliangyou 8612 (non-low cadmium hybrid rice variety) and Lianliangyou No. 1 (low cadmium version of Zhenliangyou 8612).

[0032] Experimental treatment: Four test materials were planted in two identification ponds, A and B, with different soil cadmium contents. The identification ponds were 10m long, 2m wide, and had a soil layer thickness of 30cm. Each pond had a row of 2cm diameter perforations at the bottom of the long side wall (20cm above the bottom of the external drainage ditch) and another row 30cm above the bottom. The perforations were spaced 30cm apart, with the upper and lower rows corresponding to each other. Each of the two long side walls had a 4cm diameter water inlet pipe at 10cm above the soil surface, with a 1.5cm diameter perforation at every 30cm interval. Pond A had a total soil cadmium content of 1.071mg / kg and a pH of 5.6, while Pond B had a total soil cadmium content of 1.655mg / kg and a pH of 5.51. The experiment was a randomized block design with three replicates per variety. Seedlings were manually transplanted from soft trays, with one row of 14 seedlings per replicate, transplanted individually. During the tillering stage, irrigate with shallow water of 1-3cm. At the end of the tillering stage, dry the field for 7 days. After drying the field, irrigate with water of 2-3cm and keep it for 6-8 hours. Then drain the field and dry it again. When the soil surface in the pond is dry enough that it is not muddy when stepped on, irrigate with water of 2-3cm and keep it for 6-8 hours. Repeat this cycle until the plant matures.

[0033] Sampling and testing: At maturity, some panicles of Zhenliangyou 8612 and Lianliangyou 1, as well as all panicles of the male-sterile lines Longzhen 36S and Lian 1S, were collected. After threshing, they were dried in a constant-temperature forced-air drying oven at 80℃ for 48 hours, then milled into brown rice, pulverized to 100 mesh, and digested. The cadmium content of the brown rice was determined using graphite furnace atomic absorption spectrophotometry. Although the seed setting rate of the male-sterile lines Longzhen 36S and Lian 1S was very low, the rice samples obtained after harvesting and threshing all 14 panicles were sufficient for testing. The experimental results are shown in Table 1 below.

[0034] Table 1. Cadmium content (mg / kg) of brown rice from tested varieties in soils with different cadmium contents.

[0035] Table 1 shows that the maximum cadmium content in brown rice of the low-cadmium sterile line Lian 1S and the low-cadmium variety Lian Liangyou 1 was less than 0.2 mg / kg in both soils with different cadmium contents (A and B), indicating low cadmium accumulation. The maximum cadmium content in brown rice of Longzhen 36S and Zhenliangyou 8612 was greater than 0.2 mg / kg in both soils, indicating no low cadmium accumulation, which is consistent with the actual cadmium accumulation characteristics of the samples.

[0036] Example 2 An investigation of cadmium content in brown rice and leaves of low-cadmium and non-low-cadmium varieties in soils with different cadmium contents. Experimental site: Phenotypic identification platform for low cadmium accumulation characteristics in rice, Hunan Academy of Agricultural Sciences, Chunhua Town, Changsha County. The identification pond is 40m long and 3m wide, with a soil layer thickness of 40cm. There is a row of seepage holes at the bottom of the long side of the pond wall (10cm higher than the bottom of the drainage ditch outside the pond) and 40cm from the bottom up. The holes are 2cm in diameter and spaced 20cm apart, with the upper and lower rows of seepage holes corresponding to each other. The pond is surrounded by a ditch that serves as both an inlet and outlet, and the seepage holes on the pond wall also function as inlets.

[0037] Test soils: four types of soils with different cadmium contents, namely C, D, E, and F; soil C had a total cadmium content of 0.73 mg / kg and a pH of 6.5, soil D had a total cadmium content of 1.09 mg / kg and a pH of 5.6, soil E had a total cadmium content of 1.20 mg / kg and a pH of 5.9, and soil F had a total cadmium content of 1.71 mg / kg and a pH of 5.2.

[0038] Rice varieties tested: 13 low-cadmium varieties: Zhong'anzao 7, 21LG1, 21LG2 (Xizi No. 3), Zhong'an 2, Shaoxiang 100, Anliangyou 2, Qingliansi Zhan, Qingliansimiao, Low-cadmium No. 2, Low-cadmium No. 3, Lianliangyou 1, Anyou 1, Anyou 2; Two non-low cadmium varieties: Jade Needle Fragrance and Ivory Fragrance.

[0039] Experimental treatment: Fifteen tested varieties were planted in four identification pools (C, D, E, and F) with different soil cadmium contents, arranged in a randomized block design with three replicates per variety. Seedlings were manually transplanted using trays, with one row of 14 seedlings per replicate. During the tillering stage, the field was irrigated with 1-3 cm of shallow water. At the end of tillering, the field was dried for 7 days, then re-irrigated with 2-3 cm of water for 12-14 hours. The field was then drained and dried again until the soil surface was dry enough to be easily stepped on without sticking to the ground. Then, the field was re-irrigated with 2-3 cm of water for 12-14 hours. This cycle was repeated until maturity.

[0040] Sampling and testing: Samples of rice panicles and leaves from various varieties were collected at maturity. After threshing, the rice panicles were dried, milled into brown rice, and then pulverized. Leaf samples were dried at 80℃ and then pulverized to 100 mesh. The pulverized brown rice and leaf samples were digested, and the cadmium content was determined using graphite furnace atomic absorption spectrophotometry. The experimental results are shown in Table 2 below.

[0041] Table 2. Cadmium content (mg / kg) in leaves and brown rice of different varieties in soils with different cadmium contents.

[0042] Table 2 shows that in soils with different cadmium contents, the cadmium content in brown rice and leaves of the 13 low-cadmium varieties was less than 0.2 mg / kg, indicating low cadmium accumulation. In contrast, the cadmium content in brown rice and leaves of the two non-low-cadmium varieties was greater than 0.2 mg / kg, indicating a lack of low cadmium accumulation. In soils with a total cadmium content of 0.73–1.71 mg / kg, the cadmium content in the leaves of the 13 low-cadmium varieties was generally 0.5–6.6 times that of brown rice, with an average of 3.2 times, all below 0.2 mg / kg. The cadmium content in the leaves of the two non-low-cadmium varieties was generally 0.5–1.0 times that of brown rice, with an average of 0.8 times, all above 0.2 mg / kg.

[0043] The results above show that, within a certain range of soil cadmium content, even when rice samples are unavailable, the cadmium accumulation characteristics of leaf samples can still reflect the cadmium accumulation characteristics of the variety itself.

[0044] Example 3 Investigation of cadmium content in leaves and brown rice of low-cadmium rice varieties at different test sites in the middle and lower reaches of the Yangtze River Experimental sites: 13 different experimental sites in the middle and lower reaches of the Yangtze River rice-growing area, including Changsha, Yueyang, Yiyang, Hengyang, Xiangtan, Yichun, Shangrao, Pingxiang, Tongling, Huangshan, Huangshi, Ezhou, and Fuyang (soil cadmium background value 0.30~1.71 mg / kg, pH value 5.5~8.0), see Tables 3 and 4 for details.

[0045] Experimental varieties: Group A: Lianliangyou No.1, Anyou No.1, Anyou No.2; Group B: Yexiang 100, Anliangyou No.2, Qingliansi Zhan, Qinglian Simiao, Low Cadmium No.2, Low Cadmium No.3; Group C: Zhong'anzao No.7, 21LG1, 21LG2, Zhong'an No.2; a total of 13 low cadmium early, medium and late rice varieties.

[0046] Experimental treatments: Thirteen tested varieties in three groups were planted in soil samples from different cadmium content identification ponds (15m long, 1.5m wide, and 20cm deep) at different experimental sites. Each group of varieties was planted independently, and varieties within each group were arranged in a randomized block design, with each variety replicated three times. Water-raised seedlings were manually transplanted, with 16 rows and 25 seedlings per row per replicate. Hybrid rice had 2 seedlings per seedling, while conventional rice had 3-4 seedlings per seedling. In addition to conventional wet and dry management, a slightly drier management approach was adopted, with other management practices consistent with local field production.

[0047] Sampling and testing: At maturity, samples of rice panicles and leaves from various varieties were collected. After threshing, the rice panicles were dried, milled into brown rice, and pulverized. The leaf samples were dried at 80℃ and then pulverized to 60 mesh. The pulverized brown rice and leaf samples were digested and the cadmium content was determined by graphite furnace atomic absorption spectrophotometry. The experimental results are shown in Tables 3-5 below.

[0048] Table 3. Cadmium content in leaves and brown rice of low-cadmium rice varieties at different test sites (Group A)

[0049] Note: In Table 3, " / " indicates missing data due to missing field samples; the same applies to Table 4.

[0050] Table 4. Cadmium content in leaves and brown rice of low-cadmium rice varieties at different test sites (Group B)

[0051] Table 5. Cadmium content in leaves and brown rice of low-cadmium rice varieties at different test sites (Group C)

[0052] Tables 3-5 show that, except for the Fuyang site (total soil cadmium content 1.71 mg / kg, pH 6.0), where some low-cadmium varieties had leaf cadmium content greater than 0.2 mg / kg and brown rice cadmium content less than 0.2 mg / kg, the cadmium content in both leaves and brown rice of the 13 low-cadmium varieties at other test sites (total soil cadmium content 0.30–1.25 mg / kg) was simultaneously less than 0.2 mg / kg. This indicates that when the soil cadmium content exceeds 1.7 mg / kg, the leaf cadmium content of low-cadmium varieties may be at risk of exceeding the standard, failing to accurately reflect the low cadmium accumulation characteristics of the varieties themselves.

[0053] Therefore, if leaf samples are to be used instead of rice samples to characterize the low cadmium accumulation characteristics of low cadmium varieties, there are special requirements for soil identification, namely, the total cadmium content in the soil should not exceed 1.7 mg / kg. Otherwise, the representativeness of leaf samples will be reduced, which may lead to identification errors.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for identifying the low cadmium accumulation characteristics of rice male-sterile lines, characterized in that, Includes the following steps: 1) The rice sterile line to be identified was raised to obtain rice seedlings; 2) Transplant the rice seedlings into a rain-sheltered and light-permeable identification pond, and then carry out field management until maturity; the identification pond has smooth watering and drainage. The soil in the identification pool is natural soil excavated from the topsoil of paddy fields. The total cadmium content of the soil in the identification pool is 1.0~2.0 mg / kg or 1.0~1.7 mg / kg, and the thickness of the soil in the identification pool is 0.15~0.4m. The field management includes irrigation management; The irrigation management includes: maintaining a water level of 1-3 cm in the identification pond after the rice seedlings are transplanted and before the field is dried at the end of the tillering stage; drying the field for 7-9 days at the end of the tillering stage; and cyclical management of irrigation, drainage, drying, re-irrigation, and then drainage and drying the field after the field is dried at the end of the tillering stage until the rice is harvested; maintaining a water level of 2-3 cm in the identification pond for 6-24 hours each time the field is irrigated, and drying the field until it is no longer muddy when stepped on; 3) Sampling and testing of cadmium content at maturity. When the cadmium content of the rice sample is ≤0.2mg / kg, the rice male-sterile line to be identified is determined to have low cadmium accumulation characteristics; otherwise, the rice male-sterile line to be identified does not have low cadmium accumulation characteristics. The rice samples include one or more of the following: self-pollinated full grains, cross-pollinated full grains, unfertilized empty grains, panicles, and leaves.

2. The method according to claim 1, characterized in that, The leaves in the rice sample mentioned in step 3) include leaves from any location other than axillary bud regeneration seedlings, or a mixture of leaves from multiple locations.

3. The method according to claim 1, characterized in that, The rain-proof and light-transmitting identification pool is equipped with a sun panel that provides rain protection; the identification pool is equipped with water filling and / or drainage facilities.

4. The method according to claim 1 or 3, characterized in that, The identification pool has a width of 1.5~3.0m and a length of 5~50m; the long side wall of the identification pool is provided with two rows of seepage holes, the lower row of seepage holes is level with the bottom of the identification pool, and the upper row of seepage holes is level with the top surface of the soil in the identification pool. The distance between adjacent seepage holes in the same row is 0.15~0.3m, and the diameter of each seepage hole is 2~3cm. The identification pool has a row of water inlet holes on its long side wall. The height of the water inlet holes is 0-20cm higher than the upper row of seepage holes. The distance between adjacent water inlet holes in the same row is 0.15-0.3m, and the diameter of each water inlet hole is 2-3cm.

5. The method according to claim 1, characterized in that, The cadmium content detection includes the following steps: drying the rice sample and then pulverizing it to obtain a rice powder sample; The cadmium content in the rice powder sample was determined by graphite furnace atomic absorption spectrophotometry.

6. The method according to claim 1 or 5, characterized in that, When sampling and testing cadmium content, no fewer than three biological replicates are set up for the same rice sample, and the replicate with the highest cadmium content is used to determine the low cadmium accumulation characteristic.

7. The method according to claim 1, characterized in that, The seedling raising methods described in step 1) include tray seedling throwing and / or water seedling raising.

8. The method according to claim 1, characterized in that, The method includes simultaneously identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines; when identifying the low cadmium accumulation characteristics of multiple rice male-sterile lines simultaneously, each rice male-sterile line to be identified shall be planted with no less than three biological replicates, each biological replicate shall have no less than 10 holes, and one rice seedling shall be planted in each hole.

9. The method according to claim 1, characterized in that, The method also includes planting non-cadmium-low rice sterile lines as a control.

10. The application of the method according to any one of claims 1 to 9 in the breeding of low-cadmium rice varieties.

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