Method for breeding two-line sterile line of long-grain fragrant indica rice

Abstract

The invention discloses a method for breeding a long-grain aromatic indica rice two-line sterile line. The method comprises the following steps: hybridizing a two-line sterile material HD-37S and an improved maintainer line (Taifeng B / Guangdong 8B) F3 as parents, carrying out multi-generation pedigree breeding, combining with molecular marker-assisted selection aiming at a fragrance gene fgr and a grain length gene gw7, and carrying out shuttle fertility identification in high altitude of Guangxi and Hainan region, so as to finally screen out a stable sterile line of which the pollen sterility is greater than or equal to 99.9%. According to the sterile line bred by the method, parent sources are HD-37S and (Taifeng B / Guangxi 8B) F3, and the sterile line has excellent characters that the grain length is greater than or equal to 7.0 mm, the length-width ratio is greater than or equal to 4.0, and the fragrance score is greater than or equal to 75. According to the method, efficient polymerization of long grains, fragrance and stable sterility is realized, the breeding period is short, and the selection accuracy is high.

Description

Technical Field

[0001] This invention relates to the field of rice breeding technology, and in particular to a method for breeding a two-line sterile line of long-grain aromatic indica rice. Background Technology

[0002] Two-line hybrid rice, utilizing photoperiod- and temperature-sensitive genic male-sterile lines, achieves freedom of pairing and simplifies seed production procedures, representing an important development direction in hybrid rice breeding. Meanwhile, long-grain aromatic indica rice enjoys strong market demand and significant economic benefits due to its excellent appearance and eating quality. Therefore, breeding two-line male-sterile lines that combine long grains, aroma, stable sterility, and high combining ability is crucial for developing breakthrough hybrid rice varieties.

[0003] However, in traditional breeding practices, there are significant technical bottlenecks in efficiently aggregating multiple desirable traits into a single sterile line. Traditional methods mainly rely on phenotypic selection and backcrossing, which are characterized by long breeding cycles, low efficiency, and poor selection accuracy for traits such as aroma, which are not easily judged visually, and for characteristics such as fertility transition critical temperature, which are affected by the environment. Although molecular marker-assisted selection technology offers the possibility of improving selection efficiency, how to effectively combine this technology with ecological pressure screening targeting fertility stability (e.g., using low-temperature conditions in high-altitude areas to identify the fertility transition characteristics of sterile lines) to establish an efficient and precise breeding technology system and quickly obtain long-grained, aromatic two-line sterile lines with excellent comprehensive traits remains a technical challenge that needs to be solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for obtaining a two-line male-sterile long-grain aromatic indica rice with a relatively short breeding cycle, high selection accuracy, and stable ability to obtain long grains, aroma, and excellent fertility performance.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Firstly, this invention provides a method for breeding two-line sterile lines of long-grain aromatic indica rice. This method establishes a genetic basis through specific parental hybridization, precisely selects target genes with molecular markers, and enhances fertility stability screening by combining high-altitude ecological shuttle breeding, thus constructing a highly efficient and precise breeding system. The method includes the following steps:

[0007] (1) Using the two-line sterile material HD-37S as the female parent and the improved maintainer line (Taifeng B / Guang 8B) F3 as the male parent, hybridization was carried out to obtain hybrid F1 generation seeds;

[0008] (2) Plant the F1 generation plants and allow them to self-pollinate to obtain the F2 generation segregating population;

[0009] (3) In the F2 generation population, select individual plants with long grains;

[0010] (4) The selected individual plants were subjected to pedigree selection for multiple generations, and the target individual plants were selected with the help of molecular markers.

[0011] (5) The selected strains were shuttle-planted and multiplied in high-altitude areas of Guangxi and Hainan, and the fertility conversion characteristics were identified. Stable sterile single plants with pollen sterility of not less than 99.9% and self-pollination set rate of 0% were selected.

[0012] (6) The selected stable sterile single plants were propagated by seed to obtain the long-grain aromatic indica rice two-line sterile line.

[0013] Preferably, in step (4), the marker-assisted selection is performed on the aroma gene fgr and / or the grain length gene gw7. Further, genotyping is performed using molecular markers linked to the aroma gene fgr and those linked to the grain length gene gw7 to assist in selecting individual plants carrying both the fgr and gw7 genes. Even further, the molecular markers are SSR markers or SNP markers.

[0014] Preferably, in step (3), the traits of the selected individual plants also include compact plant type and / or strong tillering ability.

[0015] Preferably, in step (5), the altitude of the high-altitude area in Guangxi is 800-1000 meters.

[0016] Preferably, in step (5), the identification of fertility conversion characteristics includes light and temperature treatment in an artificial climate chamber, wherein pollen sterility and self-pollination set rate are identified under 14.5 hours of light and 23.0°C.

[0017] Secondly, this invention provides a long-grain aromatic indica rice two-line male-sterile line. The parental sources of this male-sterile line are the two-line male-sterile material HD-37S and the improved maintainer line (Taifeng B / Guang 8B) F3, and it is characterized by having the following excellent traits: grain length ≥7.0mm, length-to-width ratio ≥4.0, aroma score ≥75 points, and pollen sterility not less than 99.9%.

[0018] Preferably, the sterile line has a self-pollination seed set rate of 0% under artificial climate conditions of 14.5 hours of light and 23.0°C.

[0019] Thirdly, the present invention provides a method for preparing the seed propagation method of the two-line sterile line of long-grain aromatic indica rice described in the second aspect.

[0020] The present invention has at least the following beneficial effects:

[0021] 1. This invention combines molecular marker-assisted selection with high-altitude ecological shuttle breeding to form a complementary breeding program. Molecular marker technology (targeting genes such as fgr and gw7) enables early and precise screening of traits that are not easily judged visually, such as aroma and grain length, significantly improving selection efficiency and accuracy. Meanwhile, shuttle planting and identification in a high-altitude, low-temperature environment provides a powerful natural pressure selection process, efficiently eliminating materials with high fertility conversion temperatures and poor stability. This ensures that the selected sterile lines possess low and stable fertility conversion characteristics (e.g., approximately 23°C), significantly reducing future seed production risks. The synergy of these two methods guarantees the accuracy and reliability of breeding at both the genotypic and phenotypic levels.

[0022] 2. The method of this invention selects the HD-37S with excellent comprehensive traits and crosses it with the specific parental combination (Taifeng B / Guang 8B) F3 which is rich in the target gene. This provides a key genetic basis for the subsequent successful aggregation of target traits such as long grains, aroma, excellent plant type and stable sterility, and is a prerequisite for the successful implementation of this technical solution.

[0023] 3. The male-sterile lines bred through the above system (such as "Guxiang S") exhibit clear and verifiable phenotypic advantages: combining long grains (grain length ≥ 7.0 mm, length-to-width ratio ≥ 4.0), significant aroma (aroma score ≥ 75 points), and highly stable sterility (pollen sterility ≥ 99.9%). These male-sterile lines demonstrate significant hybrid vigor, showing strong heterosis in crosses with multiple restorer lines. They provide invaluable core germplasm resources for cultivating high-yielding, high-quality, and aromatic breakthrough two-line hybrid rice varieties, with broad application prospects. Attached Figure Description

[0024] Figure 1 This is a pedigree chart of the breeding line “Guxiang S” (Hby015S) of long-grain aromatic indica rice, which was selected in Example 1 of this invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the principles of this invention should be included within the scope of protection of this invention.

[0026] Unless otherwise specified, the main instruments and reagents used in this invention are all conventional instruments and reagents in the art. The molecular marker detection methods involved in this invention, such as PCR amplification and electrophoresis detection methods for SSR or SNP markers, are all well-known techniques in the art and can be performed with reference to standard molecular biology laboratory manuals.

[0027] Example 1: Breeding of the long-grain aromatic indica rice two-line sterile line "Guxiang S" (Hby015S)

[0028] This embodiment fully demonstrates how to efficiently breed a long-grained, aromatic, two-line sterile line with excellent comprehensive traits through a specific combination of technologies. The breeding process and generational relationships can be found in [reference needed]. Figure 1 .

[0029] 1. Parental selection and hybridization

[0030] To establish the genetic basis for superior traits such as long grains and aroma, this invention selects a specific combination of parents. The maternal parent is the two-line sterile material HD-37S, which is a known or commercially available material in the field, characterized by good combining ability and excellent agronomic traits. The paternal parent is the improved maintainer line (Taifeng B / Guang 8B) F3, which is a known line or a line that can be obtained through conventional hybridization breeding methods, possessing excellent rice quality and slender grain shape.

[0031] In the early season of 2018, artificial hybridization was carried out at the Nanning experimental base in Guangxi. To ensure purity, the female parent HD-37S was subjected to emasculation by hot water treatment (e.g., using 43-45℃ warm water), and then pollinated with the male parent. The seeds were then bagged for isolation, and harvested after maturity to obtain true F1 generation hybrid seeds.

[0032] 2. Construction of segregating populations and early phenotypic selection

[0033] In the late season of 2018, F1 generation plants were planted, exhibiting uniform plant behavior. After self-pollination, F2 generation segregating population seeds were obtained through mixed harvesting. In the early season of 2019, approximately 2000 F2 segregating population plants were planted. To initially screen for materials possessing the target plant type and grain shape, individual plant selection was conducted during the heading to maturity stage based on the following phenotypes: ① Long grain trait: priority was given to individual plants with slender grains and a length-to-width ratio significantly greater than that of ordinary varieties; ② Superior plant type: individual plants with compact plant type, strong tillering ability, and sturdy stems were selected. Approximately 150 phenotypically superior individual plants were initially selected. This step completed the initial enrichment of easily observable traits.

[0034] 3. Molecular marker-assisted selection (MAS) and pedigree selection

[0035] To address the challenge of selecting traits such as aroma that are not easily visually determined, and to achieve precise breeding at the genotype level, molecular marker-assisted selection was performed on the aforementioned 150 F2 plants. Leaf samples were taken from each plant, and molecular markers (e.g., SSR markers RM223 and RM5436) closely linked to the major aroma gene fgr (BADH2) and the major grain length gene gw7 were used for PCR and electrophoresis detection. The method described in this invention is not limited to the specific markers mentioned above. This technique can directly identify whether a plant carries the target superior allele from its genotype, overcoming the blindness and lag of phenotypic selection. The detection results accurately screened 45 plants from the 150 plants that simultaneously carried the target fgr and gw7 genes, significantly improving selection efficiency and accuracy.

[0036] Subsequently, these 45 individual plants underwent pedigree selection for multiple generations (F3 to F6). Each generation was subject to rigorous selection for agronomic traits in the field, and molecular markers were continuously used to track target genes to ensure stable inheritance and homozygosity, eliminating lines with segregated traits or lost genes.

[0037] 4. Ecological shuttle breeding and identification of enhanced fertility stability

[0038] This is a core step in ensuring the absolute stability of sterility in the selected breeding lines and reducing production risks. Starting from the F4 generation, an ecological shuttle breeding strategy is introduced.

[0039] Shuttle operation: Superior strains selected in Nanning are transplanted to the high-altitude breeding base in Jinxiu County, Guangxi (about 1,000 meters above sea level) in summer and then grown at the Ledong base in Hainan in winter.

[0040] Identification Principle and Effect: The summer temperature at the Jinxiu base is 3-5℃ lower than that of the Nanning plain area in Guangxi, forming a natural "low-temperature pressure selection field." Fertility identification under this environment can simulate and rigorously test the fertility performance of materials at critical temperatures. All materials underwent pollen iodine staining microscopy and bagged self-pollination, implementing extremely stringent selection criteria: only individual plants with pollen sterility ≥99.9% and a bagged self-pollination seed set rate of 0% were retained.

[0041] Technical advantages: This step effectively eliminates materials with high fertility conversion temperatures and those prone to producing fertile pollen at low temperatures, thus ensuring that the final surviving sterile lines have lower fertility initiation temperatures and extremely high sterility stability. Through multiple generations (F4-F7) of shuttle screening, the fertility of the materials is thoroughly purified and enhanced.

[0042] 5. Final identification and trait determination of the superior sterile line “Gu Xiang S” (Hby015S)

[0043] The stable strains (system number Hby015S) selected through the above complete process were then subjected to final authoritative identification:

[0044] Highly stable sterility: Precise identification was conducted in an artificial climate chamber at the China National Rice Research Institute under 14.5 hours of light and 23.0℃ conditions, revealing a pollen sterility of 99.52% and a self-pollination seed set rate of 0.00%. These results clearly demonstrate that the sterility of 'Guxiang S' is highly stable and fully meets the fertility requirements for production-grade two-line male-sterile lines.

[0045] Superior rice quality: Tested by the Rice and Rice Products Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs, its polished rice grains are up to 7.2mm long, with a length-to-width ratio ≥4.0 and an aroma score ≥75. The main indicators meet the NY / T 593-2021 standard for high-quality second-grade long-grain indica rice.

[0046] Excellent agronomic traits: plant height is about 84.2cm, plant type is compact, tillering ability is strong, and overall resistance is good.

[0047] The sterile line was named "Gu Xiang S". The above identification results confirm that the method of this invention has successfully and efficiently integrated multiple excellent traits such as long grains, strong aroma, and highly stable sterility into one.

[0048] Example 2: Combining ability determination and application verification of the "Gu Xiang S" sterile line

[0049] To verify the application value of the sterile line selected in Example 1, it was used as the female parent and crossbred with several high-quality restorer lines (such as TR8221 and TR Quanxiangsi) to conduct a multi-point comparison test.

[0050] The results showed that the hybrid combinations prepared with "Gu Xiang S" generally exhibited strong heterosis. For example,

[0051] 1. The hybrid variety “Guxiang S / TR8221” achieved an average yield of 605.8 kg / mu in multi-regional trials, a 3.95% increase compared to the main control variety “Fengliangyou No. 4”. Rice quality testing showed that its main indicators met the requirements for high-quality first-class long-grain indica rice in the NY / T 593-2021 standard for edible rice varieties. 2. The hybrid variety “Guxiang S / TR Quanxiangsi” performed excellently in trials, with its rice quality also meeting the requirements for high-quality second-class long-grain indica rice in the aforementioned standard. All the above hybrids successfully maintained the slender grain shape and aroma characteristics of the parent variety 'Guxiang S' in the trials and demonstrated good stress resistance.

[0052] This result fully demonstrates that the "Guxiang S" male-sterile line has good combining ability and strong heritability of traits. The hybrids produced by it have excellent overall performance in terms of yield, quality and resistance, and have the potential to be cultivated into a high-quality and high-yield new hybrid rice variety.

[0053] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for breeding a two-line sterile line of long-grain aromatic indica rice, characterized in that, Includes the following steps: (1) Using the two-line sterile material HD-37S as the female parent and the improved maintainer line (Taifeng B / Guang 8B) F3 as the male parent, hybridization was carried out to obtain hybrid F1 generation seeds; (2) Plant the F1 generation plants and allow them to self-pollinate to obtain the F2 generation segregating population; (3) In the F2 generation population, select individual plants with long grains; (4) The selected individual plants were subjected to pedigree selection for multiple generations, and the target individual plants were selected with the help of molecular markers. (5) The selected strains were shuttle-planted and multiplied in high-altitude areas of Guangxi and Hainan, and the fertility conversion characteristics were identified. Stable sterile single plants with pollen sterility of not less than 99.9% and self-pollination set rate of 0% were selected. (6) The selected stable sterile single plants were propagated by seed to obtain the long-grain aromatic indica rice two-line sterile line.

2. The method according to claim 1, characterized in that, In step (4), the molecular marker-assisted selection is performed on the aroma gene fgr and / or the grain length gene gw7.

3. The method according to claim 2, characterized in that, Genotyping was performed using molecular markers linked to the aroma gene fgr and the grain length gene gw7 to assist in the selection of individual plants that simultaneously carry both the fgr and gw7 genes.

4. The method according to claim 3, characterized in that, The molecular marker is an SSR marker or an SNP marker.

5. The method according to claim 1, characterized in that, In step (3), the traits of the selected individual plants also include compact plant type and / or strong tillering ability.

6. The method according to claim 1, characterized in that, In step (5), the altitude of the high-altitude area in Guangxi is 800-1000 meters.

7. The method according to claim 6, characterized in that, In step (5), the identification of fertility conversion characteristics includes light and temperature treatment in an artificial climate chamber, wherein pollen sterility and self-pollination set rate are identified under 14.5 hours of light and 23.0℃ conditions.

8. A two-line sterile line for long-grain aromatic indica rice, characterized in that, Its parental sources are the two-line sterile material HD-37S and the improved maintainer line (Taifeng B / Guang 8B) F3, and it has the following characteristics: grain length ≥7.0mm, length-to-width ratio ≥4.0, fragrance score ≥75 points, and pollen sterility not less than 99.9%.

9. The long-grain aromatic indica rice two-line sterile line according to claim 8, characterized in that, The sterile line had a self-pollination set rate of 0% under artificial climate conditions of 14.5 hours of light and 23.0℃.

10. A method for preparing the two-line sterile line of long-grain aromatic indica rice as described in claim 8 or 9.

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