A high-efficiency purification method for BT-type japonica rice sterile line

Abstract

The present application relates to the technical field of rice breeding, and in particular to an efficient purification method for BT-type japonica rice sterile lines, comprising the following steps: S1, establishing paired lines; S2, removing weeds at the seedling stage and tillering stage; S3, mixed pool detection; S4, plant isolation and expansion; S5, plant purification and rejuvenation. High-quality sterile line stock for mixed-line breeding can be obtained. The present application accelerates the production and application of excellent japonica rice sterile lines through rapid cycle purification and rejuvenation, effectively solves the problems of late abortion time and fast mixing and degradation speed of BT-type japonica rice sterile lines (gametophyte sterility), and ensures high-quality breeding of parent sterile lines.

Description

Technical Field

[0001] The present application relates to the technical field of rice breeding, and more specifically, to a method for efficiently purifying a BT-type japonica rice sterile line. Background Art

[0002] The purification of high-quality parents is the core technology for efficient hybrid rice seed production and industrial development, because the sterile line is the basis of hybrid rice, and the purity of the sterile line will directly affect the purity of hybrid seeds, the full play of hybrid vigor and the service life of the hybrid combination in production.

[0003] Taking the breeding of BT-type sterile lines of hybrid japonica rice as an example, since the promotion of planting in my country, great attention has been paid to its purification and prevention of impurities and purity. The purification methods of hybrid japonica rice in related technologies mainly include: plant cycle method, four-level seed production procedure, one-selection-three-plot method, etc.

[0004] The above purification method mainly obtains paired cross plant lines through typical single plant phenotype screening, single plant paired bagging, paired cross propagation and identification. At least 4 generations (years) of purification are required for large-scale seed breeding and application. In addition, due to the difference in sporophytes between japonica rice sterile lines and indica three-line lines, they are more prone to mixed degeneration, which is not conducive to the rapid application of excellent japonica rice sterile lines in production. Therefore, a purification method for a BT-type japonica rice sterile line is provided. Summary of the invention

[0005] In order to improve the problems of long generations (years) and serious mixed degeneration in the original purification method, the present application provides a purification method for BT-type japonica rice sterile line, which reduces one generation (year) compared with the traditional method and effectively maintains the original species.

[0006] A method for purifying BT-type japonica rice sterile line comprises the following steps:

[0007] S1. Establish paired plant lines: Based on the applied sterile lines and maintenance lines, N pairs of paired plant lines are planted, each pair of paired column lines is composed of N individual plants of the parent sterile line and the parent maintenance line in equal proportions, where the value of N is 100≤N≤200;

[0008] S2. Weeding during the seedling and tillering stages: remove the obviously deformed plants in the paired plant lines during the seedling and tillering stages;

[0009] S3. Mixed pool detection: At the jointing and booting stage, take equal amount of leaves from the sterile line and maintainer line of each pair of plant lines for molecular marker detection, take m single plants as a mixed pool, and then conduct variety authenticity detection to screen the paired plant lines with the same genotype as the standard sample;

[0010] S4, plant isolation and propagation: select sterile lines with complete sterility and 100% purity for artificial assisted pollination, remove the maintainer lines in the sterile lines at maturity, harvest and sample Hainan for fertility, purity identification and test cross;

[0011] S5. Strain purification and rejuvenation:

[0012] S51, in the first season, select the excellent paired strains with fertility and purity reaching 100% identified by Hainan to isolate and expand the original seed strains;

[0013] S52, identifying the test cross hybrids, and selecting the paired lines with a hybrid seed setting rate of more than 90%;

[0014] S53, then select strains from S51 and S52, and after harvest, take samples to identify fertility and purity in Hainan, select original seed strains with purity and fertility reaching 100% and high fruit set rate of paired strain test cross hybrids, and use mixed lines to breed high-quality sterile original seeds.

[0015] Preferably, the value of n in S1 can be obtained according to the number of original seeds to be propagated, and the calculation formula is as follows:

[0016] N = the amount of sterile seeds harvested from paired plants (0.75-1.5 kg) / average seed yield per mu (150 kg) × number of plants per mu (20,000).

[0017] Preferably, the value of m in S3 is ≤50.

[0018] Preferably, the implementation method of variety authenticity detection in S3 is as follows:

[0019] First, the CTAB method was used to extract the genomic DNA from the mixed pool, and the quality of DNA extraction was tested by electrophoresis. At the same time, the DNA was quantified by ultraviolet spectrophotometer. After DNA amplification, electrophoresis was used to identify the genotype.

[0020] Preferably, the specific steps of the CTAB method are as follows, including the following steps:

[0021] 1)1) Take 1cm*1cm leaves of each strain and mix them. Grind and mix them with liquid nitrogen, then take 200±10mg and put them into a 1.5ml centrifuge tube, and add 600ul of DNA extraction solution;

[0022] 2) Place the centrifuge tube in a tissue grinder, adjust the frequency to 55 Hz, and oscillate for 90 seconds;

[0023] 3) Place the centrifuge tube in a preheated 65°C oven or water bath and maintain the temperature for 40-60 minutes, turning the centrifuge tube upside down twice during the process;

[0024] 4) Add 500 μl of chloroform to each centrifuge tube, shake and let stand at room temperature for 5-10 minutes;

[0025] 5) Transfer the centrifuge tube to a high-speed centrifuge, centrifuge at 12000 rpm for 8 min, and transfer 500 μl of the supernatant to a 1.5 ml centrifuge tube;

[0026] 6) Add an equal volume of pre-cooled anhydrous ethanol to the 1.5 ml centrifuge tube containing the supernatant, turn it upside down to mix well, and place it in a -20℃ freezer for 60 minutes;

[0027] 7) Place the 1.5 ml centrifuge tube in a high-speed centrifuge and centrifuge at 12000 rpm for 8 min;

[0028] 8) Pour off the supernatant and place in an oven at 37-65°C for 2-12 hours;

[0029] 9) Add 200 μl of double distilled water, let stand at room temperature for 1 h, and shake to mix.

[0030] Preferably, the DNA extract formula consists of the following components in weight percentage:

[0031] 1.4-1.6% CTAB, 0.11-0.13% Tris-HCl (PH8.0), 0.14-0.16% EDTA (PH8.0), 1.5-1.7% NaCl, and the balance is double distilled water.

[0032] Preferably, the gel used for electrophoresis detection of DNA extraction quality in S3 is agarose gel with a concentration of 0.8%.

[0033] Preferably, the gel used for DNA amplification electrophoresis in S3 is polyacrylamide gel with a concentration of 8%.

[0034] Preferably, the specific steps of the genotype identification method in S3 are as follows:

[0035] Amplification system: DNA 2μl, forward primer 0.4μl, reverse primer 0.4μl, 2×TSINGKE Master Mix (blue) 5μl, ddH2O to 10μl;

[0036] Reaction procedure: 94°C pre-denaturation for 5 min, (94°C denaturation for 30 s, 55-60°C annealing for 30 s, 72°C extension for 30 s) n, 72°C extension for 10 min, 15°C cooling and storage, where n is the cycle number 35;

[0037] After amplification, 8% polyacrylamide gel electrophoresis was used for typing detection.

[0038] In summary, this application has the following beneficial effects:

[0039] 1. This application mainly uses molecular biological methods to quickly obtain a batch of typical paired strains whose phenotypes and genotypes are consistent with standard samples, thereby reducing the work of single plant phenotypic screening, single plant pair bagging, paired cross identification, etc., and significantly speeding up the speed of parental cycle purification;

[0040] 2. The paired strains obtained by molecular means in this application are typical single-plant mixed samples. Compared with the traditional single-plant paired bagging method, it can effectively avoid the problems of decreased cross-pollination fruiting rate of the strains due to defects such as degeneration of the stigma of a single typical single plant and reduced pollen vitality;

[0041] 3. The screening and breeding method of the present application can effectively eliminate paired plant lines containing homogeneous varieties such as BC1, BC2, BC1F1, etc. whose plant leaf shapes are similar to those of standard samples, thereby improving the problems of late sterility time and rapid mixed degeneration of BT-type japonica rice sterile lines (gametophyte sterility), and ensuring high-quality reproduction of the parent sterile lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 49 markers for authenticity test of Shen 21A variety (NY / T1433-2014, restoration gene marker RM5629 (for high-generation homogeneity restoration);

[0043] Figure 2 A technical circuit diagram of a highly efficient purification method of BT-type japonica rice sterile line of the present invention;

[0044] Figure 3 Traditional cold storage purification method for BT-type japonica rice sterile lines. DETAILED DESCRIPTION

[0045] The following is combined with the embodiments and the attached Figure 1-3 This application is described in further detail.

[0046] Example 1

[0047] A highly efficient purification method for BT-type japonica rice sterile lines, wherein the target BT-type japonica rice sterile line is the parent sterile line Shen 21A of the main variety Shenyou 28 in Shanghai as an example;

[0048] The specific steps of its rapid purification are as follows:

[0049] S1, Paired cross expansion:

[0050] In June 2018, 20 pairs of Shen 21A parent sterile lines (maintenance lines) were planted at the Zhuangxing Comprehensive Experimental Station of Shanghai Academy of Agricultural Sciences, with 100 sterile lines and 100 maintenance lines in each pair.

[0051] S2. Remove weeds during the seedling and tillering stages:

[0052] In August 2018, weeding was carried out on the sterile lines and maintenance lines at the seedling and tillering stages, mainly removing the deformed plants, so as to ensure the growth consistency within the paired plant group.

[0053] S3, mixed pool detection:

[0054] In August 2018, leaves were taken from each plant group (sterile line / maintenance line) at the jointing and booting stage to establish mixed pools, with 50 plants as one mixed pool for variety authenticity testing, and pools with genotypes consistent with the standard samples were screened. In October 2018, 17 pairs of typical sterile lines (maintenance lines) with genotypes and phenotypes consistent with the standard samples were obtained (0.75 kg of seeds for each sterile line);

[0055] The implementation method of variety authenticity detection in S3 is as follows:

[0056] The CTAB method was used to extract the genomic DNA of the mixed pool. The quality of DNA extraction was tested by 0.8% agarose gel electrophoresis. The DNA was quantified by UV spectrophotometer. After DNA amplification, 8% polyacrylamide gel electrophoresis was used for genotyping. The identification results were referred to Figure 1 ;

[0057] The specific steps of the CTAB method are as follows:

[0058] 1)1) Take 1cm*1cm leaves of each strain and mix them. Grind and mix them with liquid nitrogen, then take 200±10mg and put them into a 1.5ml centrifuge tube, and add 600ul of DNA extraction solution;

[0059] The DNA extract is composed of the following components in weight percentage:

[0060] 1.5% CTAB, 0.121% Tris-HCl (PH 8.0), 0.149% EDTA (PH 8.0), 1.638% NaCl, and the balance was double distilled water.

[0061] 2) Place the centrifuge tube in a tissue grinder, adjust the frequency to 55 Hz, and oscillate for 90 seconds;

[0062] 3) Place the centrifuge tube in a preheated 65°C oven or water bath and maintain the temperature for 50 minutes, turning the centrifuge tube upside down twice during the process;

[0063] 4) Add 500 μl of chloroform to each centrifuge tube, shake and let stand at room temperature for 8 minutes;

[0064] 5) Transfer the centrifuge tube to a high-speed centrifuge, centrifuge at 12000 rpm for 8 min, and transfer 500 μl of the supernatant to a 1.5 ml centrifuge tube;

[0065] 6) Add an equal volume of pre-cooled anhydrous ethanol to the 1.5 ml centrifuge tube containing the supernatant, turn it upside down to mix well, and place it in a -20℃ freezer for 60 minutes;

[0066] 7) Place the 1.5 ml centrifuge tube in a high-speed centrifuge and centrifuge at 12000 rpm for 8 min;

[0067] 8) Pour off the supernatant and place in an oven at 37-65°C for 2-12 hours;

[0068] 9) Add 200 μl of double distilled water, let stand at room temperature for 1 h, and shake to mix.

[0069] The specific steps of the genotype identification method are as follows:

[0070] Amplification system: DNA 2μl, forward primer 0.4μl, reverse primer 0.4μl, 2×TSINGKE Master Mix (blue) 5μl, ddH2O to 10μl, the primer information used is as follows:

[0071]

[0072]

[0073]

[0074] Reaction procedure: 94°C pre-denaturation for 5 min, (94°C denaturation for 30 s, 55-60°C annealing for 30 s, 72°C extension for 30 s) n, 72°C extension for 10 min, 15°C cooling and storage, where n is the cycle number 35;

[0075] After amplification, 8% polyacrylamide gel electrophoresis was used for typing detection.

[0076] The above operation mainly used 48 pairs of SSR markers (NY / T1433-2014) and the restoration gene marker RM5629 (for high-generation homogeneity restoration) to detect mixed pools of different sample sizes of Shen 21A, and it was clarified that under the condition of 1 / 50 mixed pools, that is, 1 Shen 24A hybrid plant / 50 Shen 21A typical plants), differences could be shown compared with the standard samples.

[0077] S4. Isolation and propagation of strains:

[0078] In March 2019, the Shen 21A paired strains were identified for fertility and purity in Hainan, and 15 paired strains with fertility and purity of 100% were selected for cold storage. In June 2019, 5 original seed strains were selected for expansion and propagation. In September 2019, the hybrids of the Hainan sterile line test cross were identified. In October 2019, 4 original seed strains were further selected for expansion and propagation, and samples were taken in Hainan for identification in March 2020.

[0079] S5. Strain purification and rejuvenation:

[0080] S51. In June 2020, three hybrid breeding sterile stock lines of the excellent Shen 21A original stock strains with fertility and purity of 100% identified by Hainan were selected;

[0081] S52. In September and October 2020, the hybrids of the Hainan sterile lines were identified and paired lines with a hybrid fruiting rate of more than 90% were selected;

[0082] S53. Based on S51 and S52, in October 2020, 4 pairs of original seed lines were further selected for paired strain expansion, and the fertility and purity were identified in Hainan by harvesting and sampling. Three pairs of original seed lines with purity and fertility reaching 100% were selected for mixed-line breeding of high-quality sterile seed lines.

[0083] Comparative Example 1

[0084] A cold storage purification method for BT-type japonica rice sterile line is different from that in Example 1 in that the specific steps are as follows:

[0085] S1. Screening of typical individual plants of sterile line (maintainer line):

[0086] From May to August 2018, based on the applied Shen 21A sterile line (maintenance line), 40 plants of each sterile line and maintenance line were planted, and 30 typical plants of each line were selected through performance observation.

[0087] S2. Anther microscopic examination and establishment of paired crosses:

[0088] In August 2018, anther microscopy was performed on 30 typical individual plants of the selected sterile lines, and 28 completely aborted plants were screened and bagged in pairs with the maintenance line. After removing the ones with damaged bags, 23 paired crosses were obtained in October 2018.

[0089] S3, Hainan purity and fertility identification:

[0090] In March 2019, the purity and fertility of the rice were identified in Hainan, and 20 sterile lines (maintenance lines) were selected for paired crosses.

[0091] S4. Establishing paired strains and propagating them:

[0092] In June 2019, 20 pairs of the sterile line (maintenance line) of the Shen 21A parent were planted at the Zhuangxing Comprehensive Experimental Station of the Shanghai Academy of Agricultural Sciences, with 100 sterile lines and 100 maintainer lines in each pair of populations;

[0093] From July to October 2019, sterile lines and maintainer lines were removed at various growth stages, including the seedling stage, tillering stage, booting stage, and heading stage, to ensure growth consistency within the paired plant population;

[0094] 16 pairs of plant lines with good phenotypic consistency, fertility and purity were screened for artificial assisted pollination and weeded out impurities at maturity;

[0095] In October 2019, more than 0.75 kg of sterile seeds were harvested from each pair and samples were taken to Hainan for fertility and purity identification and test crossing.

[0096] S5. Strain purification and rejuvenation:

[0097] S51. In March 2020, the fertility and purity of Shen 21A paired strains were identified and tested in Hainan. 15 paired strains with fertility and purity reaching 100% were selected for cold storage, and 5 paired strains were selected in June 2020 to expand the original seed strains.

[0098] S52. In September and October 2020, the hybrids of the Hainan sterile lines were identified and paired lines with a hybrid fruiting rate of more than 90% were selected;

[0099] S53. Based on S51 and S52, in October 2020, 4 pairs of original seed lines were further selected for paired strain expansion, and the fertility and purity were identified in Hainan by harvesting and sampling. Three pairs of original seed lines with purity and fertility reaching 100% were selected for mixed-line breeding of high-quality sterile seed lines.

[0100] It can be seen from Example 1 and Comparative Example 1 that the present application provides a guarantee for the efficient purification and safe seed production of the hybrid japonica rice Shenyou 28 sterile line, and the cycle is reduced by 1 year compared with the original purification method.

[0101] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for efficiently purifying BT-type hybrid japonica rice sterile lines, characterized in that: The steps include: S1. Establish paired plant lines: Based on the applied sterile lines and maintenance lines, plant N pairs of paired plant lines, each pair of paired column lines is composed of n individual plants of the parent sterile line and the parent maintenance line in equal proportions, where the value of N is 100≤N≤200; S2. Weeding during the seedling and tillering stages: remove the obviously deformed plants in the paired plant lines during the seedling and tillering stages; S3. Mixed pool detection: At the jointing and booting stage, leaves from the sterile line and the maintainer line of each pair of plant lines are taken for molecular marker detection. M single plants are used as a mixed pool, and the value of m is ≤50. Then, the variety authenticity test is carried out to screen the paired plant lines with the same genotype as the standard sample; The implementation method of the variety authenticity detection in S3 is as follows: First, the CTAB method was used to extract the genomic DNA from the mixed pool, and the quality of the DNA extraction was tested by electrophoresis. At the same time, the DNA was quantified by ultraviolet spectrophotometer. After DNA amplification, electrophoresis was used to identify the genotype. The above operation was tested by 48 pairs of SSR markers NY / T1433-2014 and restorer gene marker RM5629 for high-generation homogeneous restorer pair Shen 21A mixed pools of different sample volumes; S4, plant isolation and propagation: select sterile lines with complete sterility and 100% purity for artificial assisted pollination, remove the maintainer lines in the sterile lines at maturity, harvest and sample Hainan for fertility, purity identification and test cross; S5. Strain purification and rejuvenation: S51, in the first season, select the excellent paired strains with fertility and purity reaching 100% identified by Hainan to isolate and expand the original seed strains; S52, identifying the test cross hybrids, and selecting the paired lines with a hybrid seed setting rate of more than 90%; S53, then select strains from S51 and S52, and after harvest, take samples to identify fertility and purity in Hainan, select original seed strains with purity and fertility reaching 100% and high fruit set rate of paired strain test cross hybrids, and use mixed lines to breed high-quality sterile original seeds.

2. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 1, characterized in that: The value of n in S1 can be obtained according to the number of original seeds to be expanded, and the calculation formula is as follows: n = the amount of sterile seeds harvested from paired plants (0.75-1.5 kg) / average seed yield per mu (150 kg) × number of plants per mu (20,000).

3. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 1, characterized in that: The specific steps of the CTAB method are as follows, comprising the following steps: 1) Take a 1cm*1cm leaf and cut it into pieces into a 2.0ml centrifuge tube. Then add a number of steel balls with a radius of 0.25cm and 750μl of DNA extraction solution into the centrifuge tube and cover the tube tightly. 2) Place the centrifuge tube in a tissue grinder, adjust the frequency to 55 Hz, and oscillate for 90 seconds; 3) Place the centrifuge tube in a preheated 65°C oven or water bath and maintain the temperature for 40-60 minutes, turning the centrifuge tube upside down twice during the process; 4) Add 500 μl of chloroform to each centrifuge tube, shake and let stand at room temperature for 5-10 minutes; 5) Transfer the centrifuge tube to a high-speed centrifuge, centrifuge at 12000 rpm for 8 min, and transfer 500 μl of the supernatant to a 1.5 ml centrifuge tube; 6) Add an equal volume of pre-cooled anhydrous ethanol to the 1.5 ml centrifuge tube containing the supernatant, turn it upside down to mix well, and place it in a -20℃ freezer for 60 minutes; 7) Place the 1.5 ml centrifuge tube in a high-speed centrifuge and centrifuge at 12000 rpm for 8 min; 8) Pour off the supernatant and place in an oven at 37-65°C for 2-12 hours; 9) Add 200 μl of double distilled water, let stand at room temperature for 1 h, and shake to mix.

4. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 3, characterized in that: The DNA extract is composed of the following components in percentage by weight: Hexadecyl-trimethyl-ammonium bromide 2%, trihydroxyaminomethane 0.121%, ethylenediaminetetraacetic acid 0.149%, sodium chloride 1.638%, and the balance is double distilled water.

5. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 1, characterized in that: The gel used for electrophoresis to detect the quality of DNA extraction in S3 was agarose gel with a concentration of 0.8%.

6. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 1, characterized in that: The gel used for DNA amplification electrophoresis in S3 is a type of polyacrylamide gel with a concentration of 8%.

7. The efficient purification method of BT type hybrid japonica rice sterile line according to claim 1, characterized in that: The specific steps of the genotype identification method in S3 are as follows: Amplification system: DNA 2μl, forward primer 0.4μl, reverse primer 0.4μl, 2×TSINGKE Master Mix (blue) 5μl, ddH2O to 10μl; Reaction procedure: 94°C pre-denaturation for 5 min, (94°C denaturation for 30 s, 55-60°C annealing for 30 s, 72°C extension for 30 s) n, 72°C extension for 10 min, 15°C cooling and storage, where n is the cycle number 35; After amplification, 8% polyacrylamide gel electrophoresis was used for typing detection.

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