Method for efficiently creating brown glume rice and application thereof

Abstract

The application belongs to the field of crop genetic breeding, and relates to a method for efficiently creating brown glume rice and application thereof. hcm A stable genetically brown glume mutant is created through radiation mutagenesis, and a molecular marker of the brown glume trait is developed hcm The recurrent parent Huazhan is used as a recurrent parent and the mutant is crossed to create brown glume rice through recurrent selection combined with molecular marker assisted selection. hcm F1 hybrid seeds are obtained by crossing the sterile line Huangling 165S and the brown glume rice, and brown glume hybrid rice seeds can be obtained by planting the F1 hybrid seeds. The brown glume trait of the mutant is introduced into the recurrent parent Huazhan and its hybrid seeds in a targeted manner, and the improved recurrent parent is used to develop hybrid rice seeds. When hybrid rice seeds are produced, the color difference between the seeds of the parents can be used to distinguish hybrid seeds and self-crossed seeds of the recurrent parent, so that the scale mixing, mixed harvesting and color selection separation of hybrid seeds can be realized, and the efficiency of hybrid rice seed production can be improved.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, and relates to the creation, pairing, and seed production application of rice restorer lines. Background Technology

[0002] In 1973, my country pioneered the successful implementation of the three-line system for hybrid rice, after which hybrid rice developed rapidly, making a significant contribution to ensuring my country's food security. However, for decades, hybrid rice seed production has still primarily relied on manual sowing, transplanting, pollination, and harvesting techniques, resulting in high production costs, high labor intensity, and low efficiency, severely hindering the further development of hybrid rice. Therefore, improving hybrid rice seed production technology and increasing efficiency has become one of the effective methods to break through the predicament of hybrid rice development. Expanding the scale of seed production is key to reducing production costs and improving seed production efficiency, while achieving full mechanization of hybrid rice seed production is the inevitable path to large-scale seed production. Among these methods, the mixed sowing method using mechanized parent lines is an effective way to achieve full mechanization of hybrid rice seed production.

[0003] Mechanized seed production based on the mixed sowing of hybrid rice parents requires that the parents have the same or similar growth period and be mixed and sown in an appropriate ratio (e.g., patent publication number CN1253472A). On this basis, different technical means are needed to separate the hybrid seeds and restorer line self-pollinated seeds at harvest, or to remove the male parent to produce only the hybrid seeds, thereby achieving full mechanization. Currently, the main technical means employed are as follows: One method involves introducing herbicide-sensitive and resistance genes into the male and female parent lines, respectively. After pollination by the male parent, herbicides are sprayed to remove it. Several institutions in my country have reported successful implementations of this technique. However, this technology sometimes requires the use of transgenic methods to introduce herbicide-resistant or sensitive genes, which carries the risk of incomplete removal of the male parent after herbicide spraying and the risk of herbicide residues in the soil.

[0004] Secondly, female-sterile materials are used as the male parent. Female-sterile materials can provide pollen, but cannot produce normal self-pollinating seeds, thus limiting the reproduction of the female-sterile materials themselves.

[0005] Third, physical methods are used to separate hybrids and self-pollinated varieties after mixed harvesting by taking advantage of the differences between the male and female parent seeds. For example, if the parents have different glumes, they can be separated by a microcomputer color sorter; or if the parents have significant differences in grain weight, the male-sterile hybrids and male parent seeds can be separated by a sieve.

[0006] Among the technical means described above, the mechanized seed production method based on the color difference of the parents requires the use of parents with obvious differences in glume color. Therefore, finding germplasm resources with glume color specificity and mining relevant molecular markers is an important foundation for using this technology. Summary of the Invention

[0007] To address the aforementioned problems, this invention proposes a method for the efficient creation of brown-hulled rice and its application. It proposes the efficient creation of brown-hulled rice materials, the development and utilization of molecular markers, and the subsequent use of hull color differences for mechanized hybrid rice seed production, providing a new germplasm resource for mechanized hybrid rice seed production.

[0008] The technical solution of this invention is implemented as follows: A method for efficiently creating brown-hulled rice, comprising the following steps: (1) After more than 3 generations of self-crossing, a pure line mutant with brown husks was obtained from the mutant with brown husks. hcm (hullcolor mutant; also known as: Ishiguro No. 1); (2) Using the pure mutant from step (1) hcm Using the restorer line Huazhan as the female parent, hybridization was carried out to obtain the F1 hybrid; (3) The F1 hybrids from step (2) are backcrossed with Huazhan. After molecular marker detection, the plants that test positive and have similar agronomic traits to Huazhan are backcrossed with Huazhan. After three or more generations of backcrossing, combined with molecular marker-assisted selection, the individual plants with brown glumes and excellent comprehensive agronomic traits are self-crossed for two generations to efficiently create excellent restorer lines with brown glumes.

[0009] The pure-line mutant in step (1) above hcm Also known as: Shihei No. 1, classified as Oryza sativa hcm, with accession number CCTCC NO: P202521, it was deposited at the China Center for Type Culture Collection on July 15, 2025, at Wuhan University, Wuhan, China.

[0010] In step (2) above, the restorer line is any existing rice variety with glumes of color to be improved (except for those that are not clearly distinguishable from brown); further, the restorer line is indica rice; the preferred restorer line is Huazhan.

[0011] The primer pairs used for the above molecular marker detection are shown in SEQ ID No. 1 and SEQ ID No. 2. The comprehensive agronomic traits refer to suitable growth period, good plant and leaf morphology, high yield, excellent rice quality, and good resistance to rice blast, etc.

[0012] The application of brown-hulled rice cultivated using the above method in the selection of superior hybrid rice with brown hull traits.

[0013] Specifically, the aforementioned brown-hulled rice was used as a restorer line.

[0014] The application steps are as follows: F1 hybrids are obtained by crossbreeding rice sterile lines and brown-hulled Huazhan improved lines. Planting F1 hybrids can yield hybrid rice with brown hulls.

[0015] When breeding superior hybrid rice, the sterile line is a rice variety with a growth period similar to or slightly shorter than that of the restorer line. The seeds of the sterile line have yellow glumes, while the seeds of the restorer line have brown glumes. When F1 hybrids are produced by crossbreeding, the hybrid rice obtained by planting F1 hybrids has brown glumes.

[0016] In the above steps, the sterile line is any existing rice variety with a growth period similar to or slightly shorter than that of the restorer line; furthermore, the sterile line is indica rice; preferably, the sterile line is Huang 165S.

[0017] This application uses the mutant of Xiangzaoxian 42. hcm and with hcm Taking the Huazhan improved line and hybrid rice (Huang 165S / Huazhan improved line) obtained as examples: (1) Using the Hunan Province's main early rice variety Xiangzaoxian 42 as material, a mutant library was created through cobalt-60 radiation, from which a mutant with brown glumes of generation M0 was screened. hcm ; (2) Using the mutant obtained in step (1) hcm After three generations of self-pollination and selection, the M3 generation pure line brown-shelled mutant was obtained. hcm ; (3) Using brown-shelled mutants hcm The male parent was used as the donor male parent, and the restorer line Huazhan was used as the female parent to cross them to obtain the F1 hybrid. (4) Backcross Huazhan as the male parent and the F1 hybrid from step (3). Detect the backcross offspring with molecular markers. Continue to backcross the offspring that are positive and have agronomic traits similar to Huazhan. After three or more generations of backcrossing, combined with molecular marker-assisted selection, self-pollinate the single plants with brown glumes and excellent comprehensive agronomic traits for two generations to efficiently create improved Huazhan lines with brown glumes. (5) Using the rice sterile line Huang 165S and the brown-hulled Huazhan improved line obtained in step (4), F1 hybrid (Huang 165S / Huazhan improved line) is obtained. F1 hybrid is planted to obtain hybrid rice with brown hulls.

[0018] The primer pairs used for molecular marker detection in step (4) above are: OsPO-Marker-F2a:TCGGCATATTGCTGGACG (SEQ ID No. 1); OsPO-Marker-R2:GCCAATATCATCCATGCTC (SEQ ID No. 2); The offspring of the above-mentioned backcrosses over multiple generations all tested positive for molecular markers and had brown glumes.

[0019] The above step (4) involves backcrossing for multiple generations, specifically three or more generations, until the backcross offspring are stable and the glumes are brown.

[0020] The ever-increasing cost of seed production has severely hampered the development of hybrid rice. Utilizing the color differences between parent seeds to create more restorer lines with superior traits can be used for mechanized seed production of hybrid rice. Combining molecular markers can further enhance this... hcm The mutant traits can be rapidly introduced into restorer lines that are widely used in production to obtain improved lines or new superior restorer lines with brown glumes, which can be used for mechanized hybrid rice seed production.

[0021] The present invention has the following beneficial effects: 1. This application created a mutant with brown glumes through radiation-induced mutagenesis. hcm Through systematic selection, mutants with stable genetic traits were obtained. hcm Using the restoration line Hua Zhan as the recurrent parent and mutant hcm Through cyclical breeding combined with marker-assisted selection, an improved Huazhan line with brown husks was obtained. This invention utilizes mutants. hcm The brown husk trait, combined with molecular markers, can be rapidly and directionally introduced into the restorer line Huazhan, effectively helping to distinguish between hybrids and restorer line self-pollinated varieties. This will further promote mechanized seed production of hybrid rice based on the color differences between parent seeds, improve seed production efficiency, reduce the production cost of hybrid rice seeds, and ensure food security.

[0022] 2. This application uses the brown glume mutant. hcm A method for breeding a brown-hulled restorer line, Huazhan, was developed by recurrent selection with the restorer line Huazhan. This method combines molecular marker detection to introduce the brown-hulled trait into the restorer line. During backcrossing, molecular testing was performed on the progeny of the previous backcross before each new backcross to ensure that plants containing the molecular marker were selected for backcrossing, thus achieving stable inheritance of the brown-hulled gene during the improvement process. The creation of this restorer line combines the brown-hulled marker with variety selection, introducing the brown-hulled marker into the restorer line and obtaining an improved restorer line with brown hulls through recurrent selection. This achieves targeted improvement of the restorer line, which helps reduce the cost and improve the efficiency of hybrid rice seed production, and provides resources for obtaining suitable mechanized seed production methods based on parent-parent mixing, mixed harvesting, and color sorting.

[0023] 3. The brown husk mutant in this invention hcmBoth the molecular marker and the method of introduction were created by the inventors of this application. hcm The brown-colored Huazhan material was screened to obtain stable new superior restorer lines. These improved restorer lines can be crossbred with widely used two-line male-sterile lines such as Long 638S and Y58S, which have similar or slightly shorter growth periods, to breed new superior hybrid rice combinations. Different ratios of restorer lines and male-sterile lines were designed for mixed planting and mechanized harvesting. Hybrids produced by crossing restorer lines and male-sterile lines exhibited the yellow husk trait of the male-sterile line, while seeds produced by self-pollination of the restorer line exhibited the brown husk trait. A color sorter was then used to screen the hybrids and restorer line self-pollinations based on the color differences between the parents, thereby improving the seed production efficiency of hybrid rice and providing important varietal support for the promotion of mechanized hybrid rice seed production.

[0024] 4. Using the brown husk trait and its molecular markers obtained by this invention, after rapidly improving and obtaining rice restorer lines, suitable sterile lines can be selected and matched to obtain superior F1 hybrids. Planting the F1 hybrids will yield hybrid rice with brown husks. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 It is a brown husk mutant. hcm The manifestations; where A: brown-shelled mutant hcm The process of color change in the glumes; B: hcm The glumes and seed coat colors of the mutant and wild-type material Xiangzaoxian 42 (WT); CG: respectively hcm Comparison of key agronomic traits between mutants and wild types, including plant height, effective panicle, panicle length, thousand-grain weight, and yield per plant.

[0027] Figure 2 The results show the molecular markers obtained based on the different bands in the electrophoresis of PCR products.

[0028] Figure 3 To utilize hcm The performance of the improved Huazhan lines obtained; where A: field performance of Huazhan before harvest; B: field performance of the improved Huazhan lines before harvest; C: performance of individual rice panicles of Huazhan and the improved Huazhan lines.

[0029] Figure 4The charts show the yield traits of Huazhan and its improved lines; where AF represents the spike length, number of effective spikes, number of spikelets per spike, seed setting rate, thousand-grain weight, and yield per plant for Huazhan and its improved lines, respectively. (* indicates p<0.05, ** indicates p<0.01, ns indicates no significant difference, the same applies below).

[0030] Figure 5 The phenotypic color phenotypes of Jingliangyou 534 and the hybrid combination (Huang 165S / Huazhan improved line) are shown.

[0031] Figure 6 The chart shows the yield traits of Jingliangyou 534 and the hybrid combination (Huang 165S / Huazhan improved line); where AF: are the spike length, number of effective spikes, number of spikelets per spike, seed setting rate, thousand-grain weight and yield per plant for Jingliangyou 534 and the hybrid combination, respectively. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] Example 1: Brown rice husk material hcm (Ishiguro No. 1) Creation Method and Phenotype The inventors used Xiangzaoxian 42, a major early rice variety promoted in Hunan Province, as material and created a mutant library through cobalt-60 radiation, from which a mutant with brown husks was screened. hcm This leads to the acquisition of a pure-line brown-shelled mutant. hcm, The specific steps are as follows: (1) Using the Hunan Province's main early rice variety Xiangzaoxian 42 as material, a mutant library was created through cobalt-60 radiation, from which a mutant with brown glumes of generation M0 was screened. hcm .

[0035] (2) Using the mutant obtained in step (1) hcm After three generations of self-pollination and selection, the M3 generation pure line brown-shelled mutant was obtained. hcm .

[0036] For brown-shell mutants hcm rice seeds hcmIt has been deposited and classified as Oryza sativa hcm, with accession number CCTCC NO: P202521. It was deposited at the China Center for Type Culture Collection on July 15, 2025, at Wuhan University, Wuhan, China.

[0037] This trait, after multiple generations of self-pollination, remained stable, and we named it Shihei No. 1. Further observation revealed that the glume color of this mutant was normal before flowering, turned distinctly brown 4 days after flowering, and by 7-10 days after flowering, the color was essentially the same as the final harvest color. Figure 1 AB); hcm Only the glumes showed a brown color, which was significantly different from the control glumes, while the seed coat color after removing the glumes was not significantly different from the wild type. Figure 1 B); Harvest period, hcm There were no significant differences between the wild type and the wild type in major agronomic traits such as plant height, effective panicles, panicle length, thousand-grain weight, and yield per plant. Figure 1 CG). Preliminary analysis of rice quality indicators shows that, hcm The chalkiness of the mutants was increased compared to the wild type (but both were less than 1%), while the other indicators showed no significant changes between the mutants and the wild type (Table 1).

[0038] Table 1 Mutants hcm Agronomic traits of wild-type WT The above results indicate that utilizing this trait will not adversely affect the main trait indicators such as yield and rice quality, and it has great value in the mechanized seed production of hybrid rice.

[0039] Example 2: hcm Development of molecular markers for brown husk trait 1. Design and synthesis of molecular markers: We designed two pairs of primers to distinguish between wild-type and mutant, amplifying a length of approximately 265 bp. The reverse sequences of these two primer pairs are identical, but the positions of their forward sequences differ. The primer sequences are as follows: The mutant primer is OsPO-Marker-F2a:tcggcatattgctggacg; Post-primer OsPO-Marker-R2:GCCAATATCATCCATGCTC; Wild-type primer OsPO-Marker-F2b:ggttcggcatatgcgact; The back primer is OsPO-Marker-R2:GCCAATATCATCCATGCTC.

[0040] 2. PCR amplification, the construction system is shown in the table below: After mixing the above reaction solutions, PCR amplification is performed. The PCR amplification program is shown in the table below: After PCR amplification, the amplification products were separated by electrophoresis on a 1% non-denaturing polyacrylamide gel, and then photographed using a gel imaging system. The results were recorded as follows: Figure 2 The results showed that, using wild-type genomic DNA as a template, the target band was amplified using the OsPO-Marker-F2b and OsPO-Marker-R2 primer pairs; hcm Using genomic DNA as a template, the OsPO-Marker-F2a and OsPO-Marker-R2 primer pairs produced the target band during amplification. Given the relatively clear amplification bands from the OsPO-Marker-F2a and OsPO-Marker-R2 primer pairs, we subsequently primarily used these primers to distinguish between mutant and wild-type sequences. In the process of introducing the brown husk trait of mutant materials into commonly used superior restorer lines, this molecular marker can be used to separate brown husk restorer lines from hybrids with normal color, improving selection efficiency.

[0041] Example 3: Creation of a brown glume restorer line for rice and selection of hybrid combinations In order to carry out applied research on the full mechanization of hybrid rice seed production based on mixed planting, mechanized harvesting, and color sorting, we designed to introduce the brown husk trait of this mutant material into a commonly used superior restorer line. During seed production, the brown husk restorer line can be easily separated from the normal-colored hybrid through color sorting.

[0042] This application discloses an application of brown husk rice created using the above method.

[0043] Using the aforementioned brown-hulled rice as a restorer line, when breeding superior hybrid rice, select a rice variety with a sterile line that has a growth period similar to or slightly shorter than that of the restorer line. The seeds of the sterile line have yellow hulls, while the seeds of the restorer line have brown hulls.

[0044] The specific operating method is as follows: (1) Using a stable brown glume mutant as the donor parent, and crossing it with the restorer line Huazhan as the male parent, the F1 hybrid was obtained. At this time, the glume of the F1 hybrid was brown.

[0045] (2) The F1 hybrid was backcrossed with Huazhan to obtain the first generation of backcrosses. Molecular marker detection was performed on the first generation of backcrosses during the seedling stage to screen out the backcross progeny containing the molecular marker of brown glume. Then, after these backcross progeny headed, single plants with agronomical traits similar to Huazhan were selected and backcrossed with Huazhan again to obtain backcross progeny. Molecular marker detection and screening were performed in the same way, and the selected progeny were then crossed with the male parent. After three consecutive generations of backcrossing, a stable Huazhan improved line with brown glume was obtained. In this way, the improvement of the restorer line Huazhan was completed quickly and efficiently. The field performance of the restorer line Huazhan before and after improvement is as follows: Figure 3 A comparison of yield traits between Huazhan and its improved lines showed that the improved Huazhan lines had significantly higher spike length, number of florets per spike, thousand-grain weight, and yield per plant than Huazhan, while there was no significant difference in the number of effective spikes and seed setting rate. Figure 4 The above results indicate that using brown glumes as a material to improve the restorer line Hua Zhan is highly effective.

[0046] (3) Using the two-line sterile rice line Huang 165S as the female parent and the Huazhan improved line as the male parent, hybridization was carried out to obtain the Huang 165S / Huazhan improved line F1 hybrid. The Huang 165S / Huazhan improved line F1 hybrid and the late-maturing mid-season rice control variety Jingliangyou 534 in Hunan Province were compared after planting and maturity. The results showed that the rice husks of the Huang 165S / Huazhan improved line F1 hybrid after harvest exhibited a typical brown color. Figure 5 Meanwhile, its spike length and yield per plant were significantly greater than those of Jingliangyou 534, while the number of spikelets per spike was the opposite; its effective spike number and thousand-grain weight were extremely significantly greater than those of Jingliangyou 534, while the thousand-grain weight was the opposite. Figure 6 The above results indicate that, after harvesting, the hybrid combination made with the improved Huazhan line as the male parent not only produces brown rice husks, but also exhibits better yield traits than the control variety.

[0047] The primer pair used for molecular marker detection in step (2) above is: OsPO-Marker-F2a:TCGGCATATTGCTGGACG (SEQ ID No. 1); OsPO-Marker-R2:GCCAATATCATCCATGCTC (SEQ ID No. 2); Given that the difference in hull color between parent seeds can be used to distinguish hybrids from restorer line self-pollinations during hybrid rice seed production, it helps improve hybrid rice seed production efficiency, reduce hybrid rice seed production costs, and ensure food security. Therefore, the creation of brown hull materials and the development and utilization of molecular markers involved in this invention have significant application value for the breeding of new varieties suitable for mechanized seed production.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently creating brown-hulled rice, characterized in that, The steps are as follows: (1) Using the Hunan Province's main early rice variety Xiangzaoxian 42 as material, a mutant library was created through cobalt-60 radiation, from which a mutant with brown glumes of generation M0 was screened. hcm Using mutants hcm After three generations of self-pollination and selection, the M3 generation pure line brown-shelled mutant was obtained. hcm ; (2) Using a stable brown-hued mutant as the donor parent, and crossing it with the restorer line Huazhan as the male parent, the F1 hybrid is obtained. At this time, the husk of the F1 hybrid is brown; the brown-hued mutant is a brown-husk mutant. hcm ; (3) Backcross the F1 hybrid with Huazhan to obtain backcross generation 1. Molecular marker detection was carried out on the backcross generation 1 material during the seedling stage. Backcross offspring with the brown glume trait molecular marker were screened out first. Then, after these backcross offspring headed out, single plants with agronomic traits similar to Huazhan were selected and backcrossed with Huazhan again to obtain backcross offspring. Molecular marker detection and screening were carried out in the same way. The screened offspring were then crossed with the male parent. After three consecutive backcrosses, a stable Huazhan improved line with brown glume was obtained. The primer pair sequences used for molecular marker detection in step (3) are shown in SEQ ID No. 1 and SEQ ID No. 2; In step (1), the pure-line mutant hcm Also known as: Shihei No. 1, classified as Oryza sativa hcm, with accession number CCTCC NO: P202521, it was deposited at the China Center for Type Culture Collection on July 15, 2025, at Wuhan University, Wuhan, China.

2. The method for efficiently creating brown-hulled rice according to claim 1, characterized in that: The agronomic traits refer to suitable growth period, good plant and leaf morphology, high yield, excellent rice quality, and good resistance to rice blast.

3. The application of the brown-hulled rice created using the method described in claim 2 in the breeding of superior hybrid rice and seed production, characterized in that, The steps are as follows: using the two-line sterile rice line Huang 165S as the female parent and the Huazhan improved line as the male parent for hybridization, to obtain the Huang 165S / Huazhan improved line F1 hybrid, and planting the F1 hybrid to obtain hybrid rice with brown husks.

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