Breeding method for rapid transfer of excellent genes of polyploid rice

Abstract

The present application belongs to the field of plant genetic breeding and genetic engineering technology, and particularly relates to a breeding method for rapid transfer of excellent genes of polyploid rice. The present application uses a rice haploid induction gene OsMATL to edit in a new type of tetraploid rice, and create a knock-out mutant of the OsMATL gene. Then, the knock-out mutant of the OsMATL gene is used as a male parent to cross with different types of homologous tetraploid rice female parents, and further create a "new" diploid rice carrying excellent genes of tetraploid rice. The rice material carries excellent alleles of tetraploid rice, and can be directly used in existing rice breeding. The method of the present application can quickly transfer excellent variations in tetraploid rice, and be applied to existing rice breeding systems.

Description

Technical Field

[0001] This invention belongs to the field of plant genetics and breeding technology and genetic engineering technology, specifically relating to a breeding method for rapid transfer of superior genes in polyploid rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops, and continuously increasing rice yield has been a long-standing goal for rice breeders. Among them, autotetraploid rice is a new polyploid germplasm obtained by chromosome doubling from diploid rice. Compared with diploid rice, autotetraploid rice has the characteristics of strong resistance, high biological yield, and obvious heterosis. Therefore, some breeders have proposed a new strategy to increase rice yield by utilizing the heterosis between subspecies of autotetraploid rice (Cai Detian, Yuan Longping, Lu Xinggui. New strategies for rice breeding in the 21st century II. Super rice breeding using the dual advantages of distant hybridization and polyploidy [J]. Acta Agronomica Sinica, 2001(01):110-116.DOI:10.3321 / j.issn:0496-3490.2001.01.019.). Studies have also found that the reduction in the number of effective panicles, the number of grains per panicle, and the decrease in the seed setting rate of tetraploid rice can limit the direct application of autotetraploid rice in breeding (Song Wenchang, Zhang Yuhua. Tetraploidization of rice and its effects on agronomic traits and nutritional components [J]. Acta Agronomica Sinica, 1992, 000(2): 137; Dai Ximei, Huang Qunce, Li Guoping, et al. Developmental characteristics of pollen in autotetraploid rice [J]. Chinese Journal of Rice Science, 2006, 20(002): 165-170. DOI: 10.3321 / j.issn: 1001-7216. 2006.02.009.).

[0003] Existing research results indicate that tetraploid rice differs from diploid rice in gene sequence and gene expression (Yu Hang, Shahid Muhammad Qasim, Li Qihang, Li Yudi, Li Cong, Lu Zijun, Wu Jinwen, Zhang Zeming, Liu Xiangdong. Production assessment and genome comparison revealed high yield potential and novel specific alleles associated with fertility and yield in neo-tetraploid rice[J]. Rice, 2020, 13(1):32.). Meanwhile, our laboratory has obtained a batch of new tetraploid rice germplasm with normal seed setting rate through years of breeding. The seed setting rate of its hybrid F1 can reach over 80%, which has been named "novel tetraploid rice".This novel tetraploid rice possesses advantages such as high fertility, strong and stable heterosis, and carries the superior allele HSP101, which is not found in diploid rice or autotetraploid rice. It holds great promise for polyploid rice breeding. (Guo Haibin, Mendrikahy JeanNestor, Xie Lei, Deng Junfeng, Lu Zijun, Wu Jinwen, Li Xiang, Shahid Muhammad Qasim, Liu Xiangdong. Transcriptome analysis of neo-tetraploid rice reveals specific differential gene expressions associated with fertility and heterosis[J]. Scientific Reports, 2017, 7(40139): 1-11; Yu Hang, Li Qihang, Li Yudi, Yang Huijing, Lu Zijun, Wu Jinwen, Zhang Zeming, Shahid Muhammad Qasim, Liu Xiangdong. Genomics analyses reveal unique classification, population structure and novel allele of neo-tetraploid rice.) Rice[J].Rice,2021,14(1):16;Liu Xiangdong, Wu Jinwen, SHAHID Muhammad Qasim. Research progress on the creation of novel tetraploid rice and its heterosis utilization[J]. Biotechnology Bulletin,2022,38(1):44-50.DOI:10.13560 / j.cnki.biotech.bull.1985.2021-0406.). Therefore, it is of great significance to discover and transfer superior genes in tetraploid rice and apply them to rice breeding.

[0004] Currently, there are no reports on the rapid and direct transfer and utilization of superior genes from tetraploid rice. However, the rice OsMATL gene has the ability to induce and produce haploid plants, showing good application potential in rice breeding. For example, Chinese invention patent CN119020519A uses a knockout mutant of both the OsMATL and OsDMP genes as the male parent. By crossing it with a female parent possessing superior traits, haploid offspring carrying the superior traits of the female parent can be obtained. However, this method requires chromosome doubling, making it difficult to rapidly obtain superior homozygous rice lines in a single step. Furthermore, OsMATL, as one of the participating genes, can work in conjunction with OsSPO11-1, OsREC8, and OsOSD1 (meiosis regulatory genes) in a multi-gene editing system to achieve the conversion of diploid cloned gametes into diploid cloned offspring, creating rice apomixis (AOP) materials (Xie En, Li Yafei, Tang Ding, Lv Yanli, Shen Yi, Cheng Zhukuan. A strategy for generating rice apomixis by gene editing[J]. Journal of Integrative Plant Biology, 2019.DOI:10.1111 / jipb.12785). Previous studies have also used OsMATL. However, OsMATL is only one of the participating genes; it needs to work synergistically with genes such as OsSPO11-1, OsREC8, and OsOSD1 to fix heterosis. In tetraploid rice, considering its unique genetic background, each chromosome has four copies. If gene editing technology is used to simultaneously knock out four genes, it would take at least 8-10 generations or 4-5 years to obtain offspring with all four allele loci being homozygous. Therefore, new breeding methods are needed to rapidly transfer superior genes from polyploid rice.

[0005] In summary, it is necessary to develop new breeding methods for the rapid transfer of superior genes in polyploid rice, so as to quickly transfer superior variations in tetraploid rice and apply the superior alleles in tetraploid rice to the existing rice breeding system. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, this invention rapidly transfers superior genes from polyploid rice, creates a new diploid rice material carrying superior genes, and applies this material to existing rice breeding.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of this invention provides a breeding method for rapid transfer of superior genes in polyploid rice, specifically: knocking out the rice haploid inducing gene OsMATL in tetraploid rice to create an OsMATL gene knockout mutant, and then using the OsMATL gene knockout mutant as the male parent and hybridizing it with a homologous tetraploid rice female parent to create a "new" diploid rice carrying the superior genes of tetraploid rice.

[0009] To rapidly transfer superior genes from tetraploid rice, this invention utilizes only one rice haploid inducible gene, OsMATL. By knocking out this gene, a homozygous knockout mutant, osmatl, is created in tetraploid rice. 4x Then, using the OsMATL gene knockout mutant as the male parent, it was hybridized with different types of homologous tetraploid rice as the female parent to create a "new" diploid rice carrying the superior alleles of tetraploid rice. Research revealed that the agronomic traits of this rice material were consistent with those of the diploid variety, but it carried the alleles of tetraploid rice.

[0010] The method of this invention can rapidly transfer superior variations in tetraploid rice. It is highly operable and technologically innovative. By rapidly creating diploid rice, the superior alleles in tetraploid rice can be directly applied to the existing rice breeding system.

[0011] Preferably, the method for creating a knockout mutant of the OsMATL gene is as follows: using the OsMATL gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, then ligating a DNA fragment containing the encoding the sgRNA sequence into a vector carrying CRISPR / Cas9, transforming rice, and thus obtaining a knockout mutant of the OsMATL gene.

[0012] More preferably, the target sites of the sgRNA sequence are target site 1 located at 290-310 bp of the OsMATL gene and / or target site 2 located at 1497-1516 bp of the OsMATL gene.

[0013] More preferably, the sgRNA sequence includes the nucleotide sequence shown in SEQ ID No. 1 and / or the nucleotide sequence shown in SEQ ID No. 2.

[0014] More preferably, the tetraploid rice includes the novel tetraploid rice Huaduo 1 and the indica-type homologous tetraploid rice Huanghuazhan-4x.

[0015] The second aspect of this invention provides the application of the breeding method for rapid transfer of superior genes in polyploid rice described in the first aspect in rice breeding.

[0016] Preferably, the rice breeding includes regulating the rice seed setting rate and regulating the rice fertility.

[0017] Preferably, the rice breeding methods include, but are not limited to, transgenic, hybrid, backcross, self-pollination, or asexual reproduction.

[0018] The third aspect of this invention provides the application of the breeding method for rapid transfer of superior genes in polyploid rice described in the first aspect in the transfer of superior genes from tetraploid rice.

[0019] The fourth aspect of this invention provides the application of the breeding method for rapid transfer of superior genes in polyploid rice described in the first aspect in the rapid reduction of polyploid rice.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention discloses a method for rapidly transferring and utilizing specific genes in tetraploid rice. Specifically, it utilizes only the rice haploid gene OsMATL in a novel tetraploid rice variety, obtaining a homozygous mutant with the target gene knocked out through gene editing technology. Then, using the OsMATL gene knockout mutant as the male parent, it is crossed with different types of tetraploid rice female parents. In the F1 generation, "new" diploid rice offspring carrying the superior genes of tetraploid rice can be obtained. This rice material carries the superior alleles of tetraploid rice, and the resulting diploid offspring are highly stable and can be directly applied to existing rice breeding. This invention directly overcomes the limitations of directly transferring specific genes in tetraploid rice and directly utilizing them in diploid rice, providing a new mutation scheme for diploid rice breeding. Attached Figure Description

[0022] Figure 1 For the mutant osmatl 4x Research on gene knockout target sequences and their phenotypic differences;

[0023] Figure 1 In the middle: Figure A shows the mutant osmatl 4x Gene knockout target sequences and mutation types; Figure BD represents the mutant osmatl. 4x Differences in plant type between wild-type Huaduo No. 1 and osmatl, where Figure B shows the plant type of wild-type Huaduo No. 1 and Figure C shows the mutant osmatl. 4x Figure D shows the plant morphology of the mutant osmatl -1. 4x Plant type diagram of -2; Figure EG represents the mutant osmatl 4x Differences in ear morphology between wild-type Huaduo 1 and osmatl, where Figure E shows the ear morphology of wild-type Huaduo 1 and Figure F shows the ear morphology of the mutant osmatl. 4x -1 ear morphology, Figure G shows the mutant osmatl4x -2 ear morphology; Figure HM represents the mutant osmatl 4x A comparison of key agronomic traits between wild-type Huaduo 1 and the mutant osmatl, where Figure H shows the comparison between wild-type Huaduo 1 and the mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of differences in seed setting rate. Figure I shows the difference between wild-type Huaduo 1 and mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of plant height. Figure J shows the difference between wild-type Huaduo 1 and the mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of differences in effective spike number. Figure K shows the differences between wild-type Huaduo 1 and the mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of differences in main spike length. Figure L shows the differences between wild-type Huaduo 1 and the mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of differences in sword leaf length. Figure F shows the differences between wild-type Huaduo 1 and the mutant osmatl. 4x -1. Mutant osmatl 4x -2 Comparison of differences in flag leaf width. In the figure above, Huaduo1 is the new tetraploid rice variety Huaduo 1. In the figure above, ** indicates a highly significant difference under the extreme condition of p < 0.01, and the error bar indicates the standard error (SE).

[0024] Figure 2 The phenotypic traits and ploidy identification results of the "new" diploid rice dhm6 and its parents;

[0025] Figure 2In the middle section: Figures AC and DF show the differences in plant architecture between the "new" diploid rice dhm6 and its autotetraploid rice Huanghuazhan-4x and original species Huanghuazhan-2x, with Bar = 20cm in the plant architecture figures; Figures DF and GF show the differences in panicle morphology between the "diploid-induced progeny" dhm6 and its autotetraploid rice Huanghuazhan-4x and original species Huanghuazhan-2x, with Bar = 5cm in the panicle morphology figures; Figure GI shows the differences between the "new" diploid rice dhm6 and its autotetraploid rice Huanghuazhan Pluripotency test results for -4x and the original Huanghuazhan-2x; Figure JO shows the comparison of differences in major agronomic traits between the "new" diploid rice dhm6 and the autotetraploid rice Huanghuazhan-4x and the original Huanghuazhan-2x. Figures J, K, L, M, N, and O represent the differences in seed setting rate, effective panicle number, panicle length, plant height, flag leaf length, and flag leaf width between the "new" diploid rice dhm6 and the autotetraploid rice Huanghuazhan-4x and the original Huanghuazhan-2x, respectively. In the above figures, ** indicates extremely significant differences under the extreme condition of p < 0.01, and the error bar indicates the standard error (SE).

[0026] Figure 3 A comparison of the differences in seedling stage and yield per plant between the new diploid rice dhm6 and its parents;

[0027] Figure 3 In the middle: Figure AC shows the difference between the new diploid rice dhm6 and the autotetraploid rice Huanghuazhan-4x and the original species Huanghuazhan-2x at the seedling stage, where Bar = 5cm in the seedling stage figure; Figure D shows the difference in yield per plant between the new diploid rice dhm6 and the autotetraploid rice Huanghuazhan-4x and the original species Huanghuazhan-2x. Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0030] Example 1: Creation of a homozygous OsMATL gene knockout mutant in a novel tetraploid rice

[0031] Compared to diploid rice, tetraploid rice exhibits stronger advantages in heterosis and resistance. Our previous research team used polyploid rice as the research material and, through resequencing and bioinformatics techniques, discovered (YuHang, Li Qihang, Li Yudi, Yang Huijing, Lu Zijun, Wu Jinwen, Zhang Zeming, Shahid Muhammad Qasim, Liu Xiangdong. Genomics analyses reveal unique classification, population structure and novel allele of neo-tetraploid rice. Rice, 2021, 14(1):16) that tetraploid rice carries variant genes not found in diploid rice. However, ploidy directly limits the direct application of these genes in ordinary diploid rice. Therefore, this embodiment utilizes gene editing and hybridization techniques to provide a breeding technique and process for rapidly transferring superior alleles from polyploid rice.

[0032] First, this study selected OsMATL (LOC_Os03g27610 / Os03g0393900) as the target gene and edited the OsMATL gene using CRISPR / Cas9 technology. Candidate target sites were selected at 290-310bp and 1497-1516bp of the OsMATL gene, respectively, and CRISPR / Cas9-based sgRNA sequences were designed [Target 1: 5'-CGCCGATTACTTCGACTGCA-3' (SEQ ID No. 1); Target 2: 5'-CGAGACCGGCAGGTACGTCG-3' (SEQ ID No. 2)]. DNA fragments containing the encoding the sgRNA sequences (SEQ ID No. 1 and SEQ ID No. 2) were ligated into a CRISPR / Cas9-carrying vector (the pYLCRISPR / Cas9 Pubi-H binary vector system provided by Academician Liu Yaoguang's laboratory at South China Agricultural University, available from Addgene). Then, the novel tetraploid rice variety Huaduo 1 was transformed using Agrobacterium-mediated transformation, and the positive transgenic plants were identified using Sanger sequencing technology, resulting in two independent transgenic knockout positive plants.

[0033] Multiple generations of testing and screening were performed on the obtained OsMATL gene-positive transformed seedlings, resulting in two homozygous mutants, both without T-DNA insertion. These mutants were named osmatl. 4x -1 and osmatl 4x -2, of which the homozygous mutant osmatl 4x -1 has a 4-base deletion of GATT at target site 1 and a 7-base deletion of AGGTACG at target site 2, which is replaced by T; while the homozygous mutant osmatl 4x -2 A 5-base CGATT deletion is present at target site 1, and a T base is inserted at target site 2. Figure 1 ).

[0034] Investigate and statistically analyze the OsMATL gene knockout homozygous mutant osmatl. 4x The study compared the wild-type Huaduo 1 mutant with wild-type Huaduo 1 in terms of plant height, flag leaf length, flag leaf width, main spike length, tiller number, and seed setting rate. The results showed that compared with wild-type Huaduo 1, the mutant osmatl... 4x -1 and osmatl 4x The main characteristic of the -2 mutant is a decrease in seed setting rate. The average seed setting rate of the mutant is only 7.83% and 7.03%, which is 43.98% and 44.78% lower than that of Huaduo No. 1, respectively. Regarding plant height, the mutant osmatl... 4x -1 and osmatl 4x The average plant height of mutants -2 was 114.48 cm and 114.95 cm, respectively, a decrease of 3.24 cm and 2.77 cm compared to Huaduo No. 1. Regarding the sword leaf length trait, the average sword leaf length of Huaduo No. 1 was 38.08 cm, while the two mutants showed a decrease of 5.65 cm and 2.62 cm, respectively. Regarding the sword leaf width trait, the average sword leaf width of Huaduo No. 1 was 2.26 cm, while the mutant osmatl... 4x -1 and osmatl 4x The flag leaf width of the -2 strain decreased by 0.06 cm and 0.13 cm, respectively. Regarding the main spike length trait, the average main spike length of Huaduo 1 was 27.05 cm, which was lower than that of the mutant osmatl. 4x -1 The main spike length is reduced by 0.20 cm, compared to the mutant osmatl. 4x -2 The main spike length increased by 1.46 cm. Regarding tiller number, Huaduo 1 had an average of 5 tillers, while the mutant osmatl... 4x The average number of tillers in the -1 mutant is 4.50. 4x The average number of tillers for a plant with a tiller size of -2 is 5.62. Figure 1 ).

[0035] Based on the above results, compared with wild-type Huaduo 1, the mutant osmatl4x -1 and osmatl 4x The main characteristic of -2 is a significant decrease in seed setting rate, with an average decrease of 44.38%. However, there are no significant differences in other agronomic traits (such as plant height, sword leaf length, sword leaf width, main spike length, and number of tillers).

[0036] Example 2: Inducing "new" diploid rice materials using hybridization technology

[0037] To investigate the mutant osmatl 4x The ability to induce diploid offspring through hybridization was studied using the mutant osmatl. 4x As the male parent, it was crossed with different types of tetraploid rice in the late season of 2022 and the early season of 2023. The resulting hybrid F1 offspring were observed and statistically analyzed to evaluate the mutant osmatl. 4x The frequency of diploid offspring produced through crossbreeding is shown in Table 1. Among the different types of tetraploid rice materials mentioned above, the autotetraploid rice Huanghuazhan-4x was developed by directly doubling the diploid rice variety Huanghuazhan through colchicine. Huaduo1 is a new type of tetraploid rice, which was developed by hybridizing the autotetraploid rice varieties Jackson-4x and 96025-4x, combining the seed setting rate trait, and through multiple generations of selection.

[0038] The research results indicate that when the novel tetraploid rice variety Huaduo 1 is used as the female parent, the mutant osmatl... 4x The induction rate of diploid offspring was 1.33%. When the indica-type autotetraploid rice Huanghuazhan-4x was used as the female parent, the mutant osmatl... 4x The induction rates of diploid offspring produced in the late season of 2022 and the early season of 2023 were 7.69% and 1.67%, respectively. These results demonstrate that the mutant osmatl... 4x Cross-pollination can produce diploid offspring, but the induction rate may vary in rice varieties with different genetic backgrounds (Table 1).

[0039] Table 1 Gene knockout mutant osmatl 4x Statistics on the frequency of diploid offspring produced by hybridization

[0040]

[0041] Note: In the above materials, Huaduo1 is the new tetraploid rice variety Huaduo 1, and Huanghuazhan-4x is an indica-type homotetraploid rice.

[0042] Example 3: Evaluation of the ploidy and application potential of new diploid rice materials

[0043] With the mutant osmatl 4x As the male parent, candidate diploid progeny were induced from the autotetraploid rice Huanghuazhan-4x in the late season of 2022 and the early season of 2023, respectively. This material was obtained using the mutant osmatl. 4x As the male parent, it was induced from the autotetraploid rice Huanghuazhan-4x and named dhm6. Figure 2 ).

[0044] To confirm that dhm6 is a mutant of osmatl 4x Diploid progeny induced from the tetraploid rice Huanghuazhan-4x as the male parent was analyzed by flow cytometry to determine the ploidy of the "diploid-induced progeny" dhm6 and its hybrid female parent Huanghuazhan-4x. The results showed that the relative intensity of the fluorescence signal on the x-axis of the flow cytometry result for the tetraploid rice Huanghuazhan-4x was around 200, while the relative intensity of the fluorescence signal on the x-axis of the diploid progeny dhm6 was 100. Simultaneously, the diploid original species of the tetraploid rice Huanghuazhan-4x was selected as a control, and the relative intensity of the fluorescence signal on the x-axis of the diploid original species Huanghuazhan-2x was found to be 100, a result consistent with the flow cytometry results of the "diploid-induced progeny" dhm6. The above test results indicate that "dhm6, the diploid induced progeny," is a diploid rice, specifically a diploid progeny induced from the mutant dhm6 by the homologous tetraploid rice Huanghuazhan-4x. Figure 2 ).

[0045] To evaluate the material characteristics of the diploid-induced progeny dhm6, two main agronomic traits were compared: the maternal tetraploid rice Huanghuazhan-4x, which produced dhm6, and its diploid parent Huanghuazhan-2x. The results showed that the diploid-induced progeny dhm6 exhibited significant phenotypic differences compared to Huanghuazhan-4x, showing a stronger tendency towards its parent species, Huanghuazhan-2x, although some differences remained. Specifically, in terms of plant height, the plant height of the diploid-induced progeny dhm6 was lower than both Huanghuazhan-4x and Huanghuazhan-2x, by 3.59 cm and 10.59 cm, respectively. Regarding blade length, the diploid-induced progeny dhm6 was longer than Huanghuazhan-4x. Figure 2 ).

[0046] In terms of flag leaf length, the diploid-induced progeny dhm6 mutant was on average 6.09 cm shorter than the autotetraploid rice Huanghuazhan-4x and 2.19 cm longer than the original Huanghuazhan-2x. In terms of flag leaf width, the diploid-induced progeny dhm6 mutant was on average 0.39 cm narrower than the autotetraploid rice Huanghuazhan-4x and 0.65 cm narrower than the original Huanghuazhan-2x. In terms of main panicle length, the diploid-induced progeny dhm6 mutant was on average 5.23 cm shorter than the autotetraploid rice Huanghuazhan-4x and 9 cm shorter than the original Huanghuazhan-2x. 1 cm; In terms of tiller number, the "diploid-induced progeny" dhm6 mutant had an average of 5 more tillers than the autotetraploid rice Huanghuazhan-4x, and 1 fewer tiller than the original Huanghuazhan-2x; In terms of seed setting rate, the "diploid-induced progeny" dhm6 mutant had an average of 37.44% higher seed setting rate than the autotetraploid rice Huanghuazhan-4x, and 26.92% lower seed setting rate than Huanghuazhan-2x; Data analysis revealed a highly significant difference in seed setting rate. From the grain shape diagram, the "diploid-induced progeny" dhm6 mutant had an average of more grains and were awnless than the autotetraploid rice Huanghuazhan-4x. Figure 3The above results indicate that the diploid-induced progeny dhm6 not only exhibits phenotypic differences from the maternal parent Huanghuazhan-4x, which induced dhm6, but also differs from the diploid parent Huanghuazhan-2x. Figure 2 and Figure 3 ).

[0047] In summary, this invention utilizes the rice haploid-inducible gene OsMATL to edit novel tetraploid rice, creating an OsMATL gene knockout mutant. This OsMATL gene knockout mutant is then used as the male parent and crossed with different types of homologous tetraploid rice female parents to create a "new" diploid rice carrying superior tetraploid rice genes. This rice material carries superior tetraploid rice alleles and can be directly applied to existing rice breeding. This method can rapidly transfer superior variations from tetraploid rice and apply them to existing rice breeding systems.

[0048] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A breeding method for rapid transfer of superior genes in polyploid rice, characterized in that, In the tetraploid rice variety Huaduo 1, the rice haploid-inducing gene OsMATL was knocked out to create an OsMATL gene knockout mutant. Then, the OsMATL gene knockout mutant was used as the male parent and crossed with the homologous tetraploid rice variety Huanghuazhan-4x as the female parent to create a diploid rice carrying the superior genes of tetraploid rice. The homologous tetraploid rice Huanghuazhan-4x was created by directly doubling the diploid rice variety Huanghuazhan using colchicine as the research material.

2. The breeding method for rapid transfer of superior genes in polyploid rice according to claim 1, characterized in that, The method for creating knockout mutants of the OsMATL gene is as follows: using the OsMATL gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, then ligating the DNA fragment containing the sgRNA sequence into a vector carrying CRISPR / Cas9, transforming rice, and thus obtaining knockout mutants of the OsMATL gene.

3. The breeding method for rapid transfer of superior genes in polyploid rice according to claim 2, characterized in that, The target sites of the sgRNA sequence are target site 1 located at 290-310 bp of the OsMATL gene and / or target site 2 located at 1497-1516 bp of the OsMATL gene.

4. The breeding method for rapid transfer of superior genes in polyploid rice according to claim 3, characterized in that, The sgRNA sequence includes the nucleotide sequence shown in SEQ ID No. 1 and / or the nucleotide sequence shown in SEQ ID No.

2.

5. The application of the breeding method for rapid transfer of superior genes in polyploid rice as described in any one of claims 1-4 in rice breeding.

6. The application according to claim 5, characterized in that, The rice breeding program includes regulating the rice seed setting rate and regulating rice fertility.

7. The breeding method for rapid transfer of superior genes in polyploid rice according to any one of claims 1-4 is used in the transfer of superior genes from tetraploid rice.

8. The breeding method for rapid transfer of superior genes in polyploid rice according to any one of claims 1-4 is used in the rapid reduction of polyploid rice.

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