Breeding method and application of fragrant soft rice three-line hybrid rice with grey white anti-counterfeiting mark

Abstract

The invention discloses a breeding method and application of fragrant soft rice three-line hybrid rice with an off-white anti-counterfeiting mark, and belongs to the technical field of rice breeding. According to the method, the problem that the taste quality and control genes thereof need to be detected and identified for multiple times during parent selection of high-quality hybrid rice is solved for the first time, consistency of the taste quality and the control genes thereof of the hybridized and matched parents is ensured, and the breeding cost is reduced; synchronous phenotype polymerization selection is carried out on an off-white endosperm marker gene representing excellent taste quality and a high combining ability, aromatic flavor and maintenance gene, so that the problem that taste quality selection is difficult to carry out on a high-quality hybridization maintenance single plant in a separation early generation is solved for the first time; in addition, soft rice marker genes with recessive grey-white endosperm are synchronously introduced into three-line hybrid rice parents, through main target character aggregation response identification, the fragrant soft rice three-line hybrid rice with grey-white markers in rice grains is successfully created for the first time, the yield is increased, and consumers can conveniently identify the authenticity of products and make brands.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of rice breeding, and particularly relates to a breeding method of soft rice three-line hybrid rice with gray-white anti-counterfeiting marks and application. BACKGROUND

[0002] The successful invention and global application of the hybrid rice breeding method have created great economic and social benefits and have obtained high evaluation at home and abroad. However, the quality, especially the eating quality, has not been effectively improved, which leads to the formation of the concept of "conventional rice has good quality, hybrid rice has poor quality, high yield is hybrid rice, and high quality is conventional rice" in the rice market. The application range of hybrid rice is not only limited, but also affects its image. At present, the overall eating quality of hybrid rice is still difficult to compare with high-quality conventional rice. Therefore, the high-quality breeding of hybrid rice, especially the excellent eating quality breeding, not only becomes a hot and difficult problem in the global rice science research in the past three decades, but also is the main attack direction of future hybrid rice breeding research.

[0003] The main reason for the difficulty and long cycle of high-quality breeding of hybrid rice is related to the special genetic characteristics of rice endosperm. Unlike conventional rice, hybrid rice seeds are already F2 generation heterozygous seeds, and most of the genes controlling rice eating quality traits are recessive, leading to the separation of eating quality genetic traits among hybrid rice grains. In other words, hybrid rice is a hybrid rice made up of a variety of rice with different eating quality, and its eating quality is lower than that of any parent selected for hybridization. It is obvious that to fundamentally prevent the genetic separation of quality traits among hybrid rice grains and to prevent the separation of internal quality or to control it within the minimum range, the connotation quality of the selected parents should be consistent, especially the Wx major gene controlling eating quality of the selected parents of hybrid rice should be equal. Therefore, to successfully breed three-line hybrid rice with excellent eating quality, not only should we successfully create a maintaining line, a sterile line and a restoring line with excellent eating quality, but also the Wx major gene controlling eating quality should be equal among the selected parents. In order to create hybrid rice varieties with excellent eating quality and soft rice, we have proposed the idea of using the heterosis of Yunnan soft rice since early times. We have adopted the method of hybrid rice parent selection, that is, "optimal-optimal, soft-soft, and fragrant-fragrant", and successfully created the fragrant soft rice hybrid rice variety Wenfu 7 with excellent eating quality and comprehensive physicochemical indicators reaching the national standard level 1. However, there are few reports on the simultaneous improvement of high-quality hybrid combination parents, that is, the simultaneous introduction of the same Wx major gene with the same eating quality into excellent maintaining materials and restoring materials, and the simultaneous identification and tracking detection of the agglomeration selection of the Wx major gene controlling eating quality, the maintaining line, the sterile line, the restoring line and the hybrid rice variety.

[0004] The main reason why it is an international problem to create a soft rice three-line hybrid rice variety with excellent flavor quality is that the created variety not only needs to be high-yielding, disease-resistant, and widely adaptable, but also needs to introduce cytoplasmic maintenance genes, restoration genes, realize the matching of three-line systems (maintainer line, sterile line, and restorer line), and integrate soft rice genes with low amylose content and aroma genes. In addition, the differences in combining ability and agronomic traits of the selected parents, especially the heterozygosity of the Wx major gene controlling the quality of soft rice and the aroma gene, and the influence of excellent flavor quality traits not only by the combined action of multiple genes, but also by the protein and fat content in the endosperm, lead to the complexity and tediousness of the procedure of creating hybrid parents, the uncertainty and difficulty. For example, if the Wx major gene and the aroma gene from different genetic backgrounds of hybrid parents (sterile line and restorer line) controlling the formation of high-quality flavor quality are not allelic, even if the selected parents have excellent flavor quality and strong aroma, it is still difficult to develop a hybrid rice variety with excellent flavor quality and strong aroma. Moreover, unlike other phenotypic agronomic traits, most of the flavor quality traits are controlled by recessive genes and have no phenotypic marker traits, which cannot be selected by sensory phenotype identification. Therefore, the allelic test of flavor quality traits genes is generally conducted by hybridization and planting identification of hybrid F1 quality and its selected parents, especially the difference in flavor quality. At present, in the creation of soft rice hybrid rice parents, in order to ensure that the Wx major gene controlling the flavor quality traits of the selected soft rice hybrid rice parents has allelic, and to minimize the blindness of hybrid parent selection, most of the different soft rice gene donors from different genetic backgrounds, which are selected as the breeding maintainer line and restorer line, are detected for the allelic test of the Wx major gene controlling the flavor quality traits. After the stable traits of the selected hybrid offspring are detected and identified, the flavor quality and its controlling gene of the selected strain material are determined. However, the problem of tedious procedure, high cost, and long cycle of soft rice hybrid rice parent selection has not been changed.

[0005] Of course, with the progress of molecular biology techniques, the directional selection of high-quality traits can be carried out by simultaneously tracking the detection of whether the Wx major gene controlling the eating quality of soft rice is allelic in the selected materials of the maintainer line and the restorer line. However, due to the reasons such as the excellent eating quality traits being controlled by multiple genes, the diversity and variability of the Wx gene controlling the eating quality of soft rice, and the lagging development of the corresponding molecular markers, and especially the fact that the directional selection cannot be completely and accurately reflected by the detection of the Wx major gene through molecular markers, the molecular marker-assisted breeding improvement of the eating quality is currently only carried out for the male sterile line or the restorer line. There is no report on the selection of the maintainer line and the restorer line with the same allelic Wx major gene controlling the eating quality of soft rice by simultaneously tracking the detection of the Wx major gene controlling the eating quality of soft rice in the selected materials of the maintainer line and the restorer line. At the same time, it is also difficult to change the problems of high cost of parent selection and complex selection procedure by means of the tracking detection of molecular markers in the laboratory. In order to reduce the large amount of work investment brought by the sensory eating quality identification, Japan and other countries have developed instruments such as the eating meter for detection, but they cannot accurately reflect the eating quality of rice. Therefore, it has been an important technical problem worldwide to explore and utilize rice resources that can directly represent the excellent eating quality traits and the controlling genes thereof, and to reduce the tedious and cumbersome detection and identification of the eating quality of parents and the controlling genes thereof.

[0006] The research on high quality breeding of hybrid rice in China has been slow, especially for the high quality hybrid rice varieties with excellent eating quality, which can be comparable to the high quality conventional rice. The reason for the long-term failure in the breeding of high quality hybrid rice varieties is the lack of sterile lines with excellent eating quality and strong combining ability. Although the eating quality of the parent or restorer line has a certain correlation with the quality of hybrid rice, the overall quality of hybrid rice, especially the eating quality, is mainly determined by the eating quality of the female parent or sterile line. In the early hybrid rice breeding in China, due to the lack of high quality rice germplasm resources with maintaining effect on cytoplasmic male sterility, it had to rely on old varieties of early rice and their derived varieties. Since the early rice varieties not only have poor appearance quality, but also lack of aroma and have poor eating quality, this is the main reason for the poor quality of early hybrid rice in China. In the new century, with the discovery and effective use of Yunnan soft rice germplasm resources with excellent eating quality, three-line sterile lines such as Yixiang 1A with excellent eating quality were created, and the overall quality of hybrid rice in China has been greatly improved through the successful grouping and use of these sterile lines. However, there is still a certain distance to breed hybrid rice varieties that can match the brand high quality rice or super high quality rice in eating quality. The main reason is that the local soft rice resources with good maintaining ability for cytoplasmic male sterility have not been discovered yet. Even if the local soft rice resources with good maintaining ability for cytoplasmic male sterility are found, due to the poor combining ability and agronomic traits, they cannot be directly used as maintaining materials to breed sterile lines. Therefore, it is necessary to introduce the excellent maintaining ability genes and high combining ability genes, Wx major genes for controlling excellent eating quality, aroma genes and other excellent agronomic trait genes through hybridization, and then through aggregation selection, generation separation and selection of soft rice lines with excellent eating quality, and further breeding of sterile lines. Especially in the process of generation separation and purification of the main target traits, in order to improve the accuracy of selection, on the basis of ensuring excellent agronomic traits, not only the maintaining ability and combining ability need to be determined, but also the aggregation of Wx major genes for controlling excellent eating quality, aroma genes, maintaining ability genes and high combining ability genes need to be tested. In addition, the maintaining genes, combining ability genes and eating quality genes cannot be determined by sensory phenotype, but need to be determined by hybridization identification or molecular marker detection for comprehensive judgment and selection of maintaining lines with purebred maintaining genes, high combining ability genes and excellent eating quality trait genes and other excellent agronomic trait genes. Especially in the maintaining hybrid separation generation, not only the appearance of the rice grains obtained from the single plant is basically transparent and consistent, but also the seed quantity is extremely small due to the selection of lines. In addition, the excellent eating quality trait is the result of the comprehensive action of multiple genes, which makes it difficult to accurately detect and identify, which further increases the unpredictability and difficulty of selecting maintaining lines with excellent eating quality. Therefore, the creation of soft rice lines with strong combining ability, excellent eating quality and strong aroma is still a technical problem to be solved urgently in the world today.

[0007] Heterosis utilization is still one of the most effective measures to improve rice yield at present. With the improvement of hybrid rice quality, the application area of high-quality hybrid rice will be more and more extensive. However, the appearance of various types of rice in the world, including high-quality hybrid rice and brand-packaged fragrant rice, is almost transparent, the fragrance is similar, and the human senses are not easy to identify. In addition, the market sales price is high, which provides an opportunity for illegal addition of synthetic flavor and other counterfeit fragrant rice. Counterfeit and inferior fragrant rice products are not uncommon, which has disturbed the fragrant rice market and infringed the rights and interests of consumers. Therefore, how to use biological breeding technology to mark the high-quality fragrant hybrid rice with obvious marks so that consumers can easily distinguish and maintain the rights and interests of consumers and promote the healthy development of the fragrant soft rice industry in China is an important technical problem to be solved. SUMMARY

[0008] The purpose of the present application is to provide a breeding method and application of fragrant soft rice three-line hybrid rice with gray-white anti-counterfeiting marks. To solve the problems in the prior art that multiple detection and identification of the taste quality and control genes of the selected parents are required to ensure the consistent taste quality of the hybrid selected parents and the control of the alleles of the genes, the taste quality and control genes of the selected parents are difficult to select in the earliest separation generation, the yield of high-quality fragrant soft rice is low, the market sales price is high, and the high-quality fragrant soft rice is easy to be infringed by counterfeit and inferior products.

[0009] In a first aspect, the present application provides a breeding method of a soft rice three-line hybrid rice with gray-white anti-fake mark, comprising the following steps: S1, providing B donor varieties Ⅲ with maintaining ability and having recessive gray-white endosperm marker genes and strong rice aroma genes, and C donor varieties Ⅳ with restoring ability and having recessive gray-white endosperm marker genes and strong rice aroma genes, respectively; S2, selecting D excellent three-line maintainer lines Ⅴ to perform incomplete diallel cross with the donor varieties Ⅲ, to obtain hybrid seed of the hybrid lines with the recessive gray-white endosperm marker genes, strong rice aroma genes, high combining ability genes and strong maintaining ability genes, and through field advantage identification, the hybrid seed of the hybrid combination with the highest yield and strong rice aroma is selected for planting and generation, and at the same time, the target trait is simultaneously selected for phenotype aggregation, and through multiple generations of self-crossing and separation selection, E soft rice maintaining lines Ⅶ with recessive gray-white endosperm marker and strong rice aroma are obtained; selecting an excellent three-line sterile line Ⅷ as a female parent to cross with the soft rice maintaining line Ⅶ, and planting and bagging self-crossing of the hybrid seed of the hybrid combination, and taking the male parent of the hybrid combination with complete failure of cytoplasmic male sterility or complete abortion and 0% self-crossing seed setting rate as a recurrent parent, to perform multiple generations of backcrossing and nuclear replacement, to obtain F soft rice three-line sterile lines Ⅸ with recessive gray-white endosperm marker and strong rice aroma; S3, selecting G excellent restoring lines Ⅵ to perform incomplete diallel cross with the donor varieties Ⅳ, to obtain hybrid seed of the hybrid lines with the recessive gray-white endosperm marker genes, strong rice aroma genes, high combining ability genes and strong restoring ability genes, and through field advantage identification, the hybrid combination with the highest yield and strong rice aroma is selected for planting and generation, and at the same time, the target trait is simultaneously selected for phenotype aggregation, and through multiple generations of self-crossing and separation selection, H soft rice three-line restoring lines Ⅹ with the same taste quality as the soft rice three-line sterile lines Ⅸ, control genes, high combining ability, strong restoring ability, recessive gray-white endosperm marker and strong rice aroma are obtained; S4, performing incomplete diallel cross on the soft rice three-line sterile lines Ⅸ and the soft rice three-line restoring lines Ⅹ, and performing target trait aggregation response identification on the hybrid seed of the hybrid combination, to obtain a soft rice three-line hybrid rice variety Ⅺ with gray-white anti-fake mark of the rice grain with excellent taste quality and strong rice aroma; wherein, B, C, D, E, F, G and H are positive integers.

[0010] The hybrid in the present application is a conventional rice hybridization technology, and the key points are: first, according to the biological characteristics of the male parent and the female parent, cultivation technical measures are taken to make the male parent and the female parent bloom at the same time; second, the pollen of the male parent is transmitted to the stigma of the female parent through artificial emasculation of the female parent (when the female parent is a sterile line, artificial emasculation is not needed) and artificial pollination, to perform hybridization pollination and obtain hybrid rice seeds.

[0011] In this invention, the inventors discovered that by introducing a gene for the trait of grayish-white endosperm, which directly characterizes excellent eating quality, into a superior maintainer line, and by effectively fusing the marker gene, the gene for strong aroma, the gene for good retention of cytoplasmic sterility, the gene for high combining ability, and other genes for excellent agronomic traits, simultaneous phenotypic aggregation selection was conducted with grayish-white endosperm marker, strong aroma, good retention ability, and high combining ability as the main target traits. This successfully created a maintainer line and its sterile line for fragrant soft rice with excellent eating quality, grayish-white marker in the grains, and strong aroma. Simultaneously, using the same aggregation selection method, a Wx major-effect gene controlling the formation of excellent eating quality was created, exhibiting strong restoring ability, high combining ability, and excellent eating quality, grayish-white marker in the grains, and strong aroma. This achieved a three-line system. Subsequently, by employing the main target aggregation response identification method, a three-line hybrid rice variety with excellent eating quality, grayish-white marker in the grains, and strong aroma was successfully bred. This hybrid rice parent selection method allows for targeted selection of single plants with superior eating quality while maintaining the F1 generation of hybrid offspring. It also eliminates the need for identifying the eating quality of superior strains and detecting the alleles of the major Wx genes controlling eating quality. The parent selection is highly targeted, the hybrid parent pairing is precise, the selection procedure is simpler and clearer, and the selection cost is reduced. At the same time, the bred varieties have excellent eating quality, high yield, and the rice grains have a gray-white mark, which helps consumers identify the authenticity of the product and protects independent intellectual property rights.

[0012] In some implementation schemes, the breeding methods for donor varieties III and IV in step S1 include: using superior three-line sterile line I as the female parent, hybridizing it with A aromatic soft rice germplasm resources II that have recessive gray-white endosperm markers; planting and bagging the resulting hybrid seeds for self-pollination; selecting the male parent of the hybrid combination with complete pollen abortion, a self-pollination seed setting rate of 0%, good plant and leaf morphology, and strong aroma, which is the donor variety III; and selecting the male parent of the hybrid combination with normal pollen, a self-pollination seed setting rate of over 90%, good plant and leaf morphology, strong aroma, and a different genetic background from donor variety III, which is the donor variety IV; where A is a positive integer.

[0013] In some implementation schemes, the aromatic soft rice germplasm resource II refers to rice varieties and their derivatives that are derived from domestic or foreign sources and whose endosperm contains a recessive gray-white marker gene (g) and a recessive aroma-rich gene (s).

[0014] In some preferred embodiments, the soft rice germplasm of type Ⅱ includes one or more of WGR11-682, WGR11-726, WGR11-735, WGR11-748, WGR11-760, WGR11-767, WGR11-770, WGR11-784, WGR14-636, WGR14-642, WGR14-646, WGR14-657, WGR14-668, WGR14-766, WGR14-774, WGR14-785, WGR14-792, WGR14-798, WGR15-428, WGR15-443, WGR15-447, WGR15-459, WGR15-466, WGR16-776, WGR16-786, WGR16-789, WGR16-792, and WGR16-804.

[0015] In some more preferred embodiments, the donor variety Ⅲ includes at least one of WGR14-766, WGR14-798, and WGR15-466; the donor variety Ⅳ includes at least one of WGR16-786, WGR11-726, and WGR16-804.

[0016] In some embodiments, the elite three-line sterile line Ⅰ includes at least one of Wenxiang 28A and Yixiang 1A.

[0017] In some embodiments, in step S2, the synchronous phenotypic aggregation selection of the target traits specifically includes: in the process of generation advancement, taking the recessive gray-white endosperm marker trait as a selection marker for excellent eating quality, taking the recessive gray-white endosperm marker trait and strong aroma as the main target traits, and combining the shelling observation and the chewing of brown rice, the plants with excellent eating quality and strong aroma are selected from F1 generation by phenotypic aggregation line selection.

[0018] In the present application, the recessive gray-white endosperm marker is taken as a representation of excellent eating quality trait, the shelling observation and the chewing of brown rice are adopted, and without the aid of eating quality identification, molecular markers, and eating meter, etc., the rice grains with the recessive gray-white endosperm marker and strong aroma (F2 seeds) are selected from the seeds of the F1 generation plants, and are planted for generation advancement to other agronomic traits, so that the rice grains with the gray-white endosperm marker and strong aroma, which have the traits of excellent eating quality and strong aroma, are selected, and the soft rice of type Ⅱ with the gray-white endosperm marker and strong aroma is obtained. Since the gray-white endosperm marker and the strong aroma both belong to traits controlled by recessive genes, once the phenotypic traits are aggregated and expressed, the genotype is stable and pure, and will not disappear in the generation separation.

[0019] In some embodiments, in step S2, the number of generations for the multiple generations of self-intersection and separation selection is 6-8 generations; and the number of generations for the multiple generations of backcrossing and nuclear replacement is 6-8 generations.

[0020] It can be understood that the number of generations of multiple generations of self-intersection separation selection, multiple generations of backcrossing and nuclear replacement can be routinely selected according to actual use needs, as long as a sterile line with stable agronomic traits is obtained. For example, in the present application, the number of generations is preferably 6-8 generations.

[0021] In some embodiments, in step S2, the excellent three-line maintainer line VII includes at least one of Wenxiang 24B, Wenxiang 28B, Zhuangxiang 2501B, Yixiang 1B, and Zhong 9B.

[0022] In some embodiments, in step S2, the aroma soft rice maintainer line VII includes at least one of WGM19-324, WGM19-332, WGM19-338, WGM19-342, WGM19-347, and WGM19-350.

[0023] In some embodiments, in step S2, the excellent three-line sterile line VIII includes Wenxiang 28A.

[0024] In some embodiments, in step S2, the aroma soft rice three-line sterile line IX includes at least one of WGA21-324, WGA21-332, WGA21-338, WGA21-342, and WGA21-347.

[0025] In some embodiments, in step S3, the synchronous phenotype aggregation selection of target traits specifically includes: during the generation process, taking the recessive gray-white endosperm marker trait as a selection marker for excellent eating quality, taking the recessive gray-white endosperm marker trait and strong aroma as the main target traits, combining shelling observation and chewing brown rice method, and performing phenotype aggregation line selection on F1 generation for excellent eating quality and strong aroma traits.

[0026] In the present application, the recessive gray-white endosperm marker is taken as a representation of excellent eating quality traits, and a germplasm resource variety with this trait is taken as an excellent eating quality gene donor variety. By hybridization and integration of other excellent traits, as long as this trait is taken as a phenotypic selection marker, the control of this eating quality trait gene will not be lost due to gene recombination and trait separation selection during generation breeding, and there is no need for auxiliary detection such as eating quality identification, molecular markers, and eating quality meter. The selection of the excellent hybrid mating parents in the generation line selection process can be accurately grasped by this phenotypic trait. At the same time, as long as the excellent hybrid mating parents have this phenotypic trait, it indicates that the eating quality of the two is consistent and the control genes are equal.

[0027] In some embodiments, in step S3, the number of generations of multiple generations of self-intersection separation selection is 6-8 generations.

[0028] It can be understood that the number of generations of the multiple generations of self-intersection separation selection can be routinely selected according to actual use needs, as long as the restorer lines can be obtained. For example, in the present application, the number of generations is preferably 6-8 generations.

[0029] In some embodiments, in step S3, the excellent restorer line VI includes at least one of Wenhu 206, Wenhu 247, Wenhu 305, Wenhu 324, Wenhu 927, and Minghu 63, Yangdao 6, Mianhu 725, Huazhan, Yihu 1577.

[0030] In some embodiments, in step S3, the soft rice three-line restorer line X of the aroma type includes at least one of WGR19-288, WGR19-297, WGR19-302, WGR19-308, WGR19-312, WGR19-316, and WGR19-320.

[0031] In some embodiments, in step S4, the target trait aggregation response identification specifically includes at least one of yield identification, adaptability identification, resistance identification, physicochemical quality identification, and sensory taste quality identification.

[0032] In some embodiments, in step S4, the amylose content of the soft rice three-line hybrid rice variety XI of the aroma type is 9.6-10.3%, the gel consistency is 89.4-95.8 mm, the alkali digestion value is 6.2-6.8, the sensory taste score is 94.8-96.4 points, and the aroma score is 94.6-96.7 points; the gray-white anti-counterfeiting marker trait in the soft rice three-line hybrid rice variety XI of the aroma type is controlled by a single recessive gene, is not affected by the environment, and can be stably inherited; and the soft rice three-line hybrid rice variety XI of the aroma type includes Wenbaiyou 9288.

[0033] In the present application, the soft rice three-line hybrid rice variety XI of the aroma type has the following taste quality characteristics after being cooked into rice: pleasant aroma, soft and refreshing, rice grains are crystal clear and have elasticity and luster, gelatinization is not sticky, does not retrograde and harden after cooling, and can be eaten cold or hot.

[0034] In a second aspect, the present application provides an application of the soft rice three-line hybrid rice breeding method with a gray-white anti-counterfeiting marker of any one of the above in rice breeding.

[0035] Compared with the prior art, the present application has the following beneficial effects: 1. The present application solves the problem of detecting and identifying the taste quality and its control gene multiple times for the selection of hybrid rice parent lines for the first time, ensures the consistency of the taste quality and its control gene alleles of the hybrid parent lines, and reduces the breeding cost; 2、The application first synchronously phenotypic polymerization selection of the gray-white endosperm marker gene with excellent eating quality and high combining ability, strong flavor and maintenance genes, and first solves the problem that high-quality hybrid maintenance single plants are difficult to select for eating quality in early generation separation, and the quality selection target is strong; 3、The application synchronously introduces the recessive gray-white endosperm soft rice marker gene into three-line hybrid rice parents, and successfully creates the three-line hybrid rice with gray-white marker and aromatic soft rice by main target trait aggregation response identification, improves yield, and is beneficial to consumer identification of product authenticity and brand building. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The application is a breeding method flow chart of the three-line hybrid rice with gray-white anti-fake marker and aromatic soft rice. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments of the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the application.

[0038] The experimental methods not specified in the embodiments are usually performed according to conventional conditions and the conditions described in the manual, or according to the conditions suggested by the manufacturer, and the general equipment, materials, reagents, etc. used are commercially available, unless otherwise specified.

[0039] If not specifically indicated, the hybridization technology used in the embodiments is the conventional hybridization technology known to those skilled in the art.

[0040] In the application, the rice making for eating quality identification and the sensory eating identification refer to the national standard “Grain and Oil Inspection Rice, Rice Cooking and Eating Quality Sensory Evaluation Method” (GB / T 15682-2008), the rice quality physicochemical index analysis is performed according to the industry standard “Edible Rice Variety Quality” (NY / T593-2002), and the rice aroma quality identification refers to the industry standard “Aromatic Rice NY / T596-2002”.

[0041] The rice varieties Wenhou 206, Wenhou 247, Wenhou 305, Wenhou 324 and Wenhou 927 involved in the following examples are fragrant soft rice restorer lines independently developed by the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences, which have passed the national variety right authorization protection, are preserved in the Germplasm Bank of the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences and are introduced from the National Crop Germplasm Resource Library of the Chinese Academy of Agricultural Sciences; The excellent three-line maintainer lines Wenxiang 24B, Wenxiang 28B and Zhuangxiang 2501B are three-line maintainer lines selected by the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences; Wenxiang 28A is a three-line sterile line selected by the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences, which has passed the national variety right authorization protection, is preserved in the Germplasm Bank of the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences and is introduced from the National Crop Germplasm Resource Library of the Chinese Academy of Agricultural Sciences; The restorer lines Minghui 63, Yangdao 6, Mianhui 725, Huazhan and Yihui 1577 are restorer lines selected by the Sanming Academy of Agricultural Sciences in Fujian Province, the Yangzhou Academy of Agricultural Sciences in Jiangsu Province, the Mianyang Academy of Agricultural Sciences in Sichuan Province, the Chinese Academy of Rice Research and the Yibin Academy of Agricultural Sciences in Sichuan Province, respectively, and can be introduced from the National Crop Germplasm Resource Library Seed Bank of the Chinese Academy of Agricultural Sciences or each breeding unit; Zhong 9B is a maintainer line selected by the Chinese Academy of Rice Research and can be introduced from the National Crop Germplasm Resource Library Seed Bank of the Chinese Academy of Agricultural Sciences and the breeding unit; Yixiang 1B and Yixiang 1A are three-line maintainer lines and their sterile lines selected by the Yibin Academy of Agricultural Sciences in Sichuan Province and can be introduced from the National Crop Germplasm Resource Library Seed Bank of the Chinese Academy of Agricultural Sciences and the breeding unit. The control variety Ne 6 You 5182 in the Yunnan Provincial Hybrid Indica Rice Regional Test is a fragrant three-line hybrid rice variety selected by the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences and the Neijiang Hybrid Rice Science and Technology Development Center in Sichuan Province in cooperation, which is preserved in the Germplasm Bank of the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences and can also be introduced from the National Crop Germplasm Resource Library Seed Bank of the Chinese Academy of Agricultural Sciences.

[0042] WGR11-682, WGR11-726, WGR11-735, WGR11-748, WGR11-760, WGR11-767, WGR11-770, WGR11-784, WGR14-636, WGR14-642, WGR14-646, WGR14-657, WGR14-668, WGR14-766, WGR14-774, WGR14-785, WGR14-792, WGR14-798, WGR15-428, WGR15-443, WGR15-447, WGR15-459, WGR15-466, WGR16-776, WGR16-786, WGR16-789, WGR16-792, WGR16-804, etc. 28 test materials are Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences through the research of local soft rice germplasm resources, screening out Haomulv (preserved number: CCTCC NO: P202407) with gray endosperm marker traits, and crossing Haomulv with Dianrui 408, Dianlong 201, Wenhua No. 2, etc. After crossing and screening strains and hybrid rice parents, the specific method is as follows: materials WGR11-682, WGR11-726, WGR11-735, WGR11-748, WGR11-760, WGR11-767, WGR11-770, WGR11-784 are single plant WR05-825 selected by hybridization of Haomulv as female parent and conventional aromatic soft rice variety Dianrui 408 in 2005, and aromatic soft rice strain material with gray endosperm marker is screened out by hybridization and separation of high recovery force and strong combining ability aromatic soft rice restorer line Wenhu 206 in 2011; materials WGR14-636, WGR14-642, WGR14-646, WGR14-657, WGR14-668 are single plant WR05-825 selected by hybridization of Haomulv as female parent and conventional aromatic soft rice variety Dianrui 408 in 2005, and aromatic soft rice strain material with gray endosperm marker is screened out by hybridization and separation of high combining ability aromatic hybrid rice restorer line Haoxiang 24B in 2014; materials WGR14-766, WGR14-774, WGR14-785, WGR14-792, WGR14-798 are single plant WR05-825 selected by hybridization of Haomulv as female parent and conventional aromatic soft rice variety Dianrui 408 in 2005, and aromatic soft rice strain material with gray endosperm marker is screened out by hybridization and separation of high combining ability aromatic hybrid rice restorer line Wenx 28B in 2014;The material WGR15-428, WGR15-443, WGR15-447, WGR15-459, WGR15-466 is a single plant WR05-442 selected from a cross between Gaomulv as a female parent and a conventional soft rice variety Wen Diao 2 in 2005, and then a cross between the high combining ability soft rice hybrid rice maintainer Zhuzhang 2501B in 2015 to screen a soft rice variety with gray-white endosperm marker; the material WGR16-776, WGR16-786, WGR16-789, WGR16-792, WGR16-804 is a single plant WR05-228 selected from a cross between Gaomulv as a female parent and a conventional soft rice variety Dianlong 201 in 2005, and then a cross between the high combining ability soft rice hybrid rice restorer Wen Hui 242 in 2016 to screen a soft rice variety with gray-white endosperm marker.

[0043] The above-mentioned screened soft rice variety with recessive gray-white endosperm marker is preserved in the Germplasm Bank of the Wenshan Zhuang and Miao Autonomous Prefecture Academy of Agricultural Sciences, and the public can obtain it from the Germplasm Bank.

[0044] The following examples are carried out according to the breeding method in Figure 1 .

[0045] Example 1 Screening of donor variety III with maintaining ability and recessive gray-white endosperm marker gene and strong aroma gene and donor variety IV with restoring ability and recessive gray-white endosperm marker gene and strong aroma gene The example takes Wenxiang 28A and Yixiang 1A as excellent three-line sterile line I (cytoplasmic male sterile line), and 28 rice materials WGR11-682, WGR11-726, WGR11-735, WGR11-748, WGR11-760, WGR11-767, WGR11-770, WGR11-784, WGR14-636, WGR14-642, WGR14-646, WGR14-657, WGR14-668, WGR14-766, WGR14-774, WGR14-785, WGR14-792, WGR14-798, WGR15-428, WGR15-443, WGR15-447, WGR15-459, WGR15-466, WGR16-776, WGR16-786, WGR16-789, WGR16-792, WGR16-804 with recessive gray-white endosperm marker as germplasm resources II of aromatic soft rice with recessive gray-white endosperm marker, to screen donor varieties III with maintaining ability and recessive gray-white endosperm marker gene (g) and strong aroma gene (s) and donor varieties IV with restoring ability and recessive gray-white endosperm marker gene (g) and strong aroma gene (s), respectively. The specific implementation is as follows: In March 2018, in the experimental base of Wenshan State Agricultural Academy in Sanya City, Hainan Province, Wenxiang 28A and Yixiang 1A were used as female parents, and 28 materials such as WGR11-682, WGR11-726, WGR11-735, WGR11-748, WGR11-760, WGR11-767, WGR11-770, WGR11-784, WGR14-636, WGR14-642, WGR14-646, WGR14-657, WGR14-668, WGR14-766, WGR14-774, WGR14-785, WGR14-792, WGR14-798, WGR15-428, WGR15-443, WGR15-447, WGR15-459, WGR15-466, WGR16-776, WGR16-786, WGR16-789, WGR16-792 and WGR16-804 were used as male parents to carry out incomplete diallel cross grouping,obtained 56 hybrid combinations of seeds of Wenxiang 28A x WGR11-682, Wenxiang 28A x WGR11-726, Wenxiang 28A x WGR11-735, Wenxiang 28A x WGR11-748, Wenxiang 28A x WGR11-760, Wenxiang 28A x WGR11-767, Wenxiang 28A x WGR11-770, Wenxiang 28A x WGR11-784, Wenxiang 28A x WGR14-636, Wenxiang 28A x WGR14-642, Wenxiang 28A x WGR14-646, Wenxiang 28A x WGR14-657, Wenxiang 28A x WGR14-668, Wenxiang 28A x WGR14-766, Wenxiang 28A x WGR14-774, Wenxiang 28A x WGR14-785, Wenxiang 28A x WGR14-792, Wenxiang 28A x WGR14-798, Wenxiang 28A x WGR15-428, Wenxiang 28A x WGR15-443, Wenxiang 28A x WGR15-447, Wenxiang 28A x WGR15-459, Wenxiang 28A x WGR15-466, Wenxiang 28A x WGR16-776, Wenxiang 28A x WGR16-786, Wenxiang 28A x WGR16-789, Wenxiang 28A x WGR16-792, Wenxiang 28A x WGR16-804, Yixiang 1A x WGR11-682, Yixiang 1A x WGR11-726, Yixiang 1A x WGR11-735, Yixiang 1A x WGR11-748, Yixiang 1A x WGR11-760, Yixiang 1A x WGR11-767, Yixiang 1A x WGR11-770, Yixiang 1A x WGR11-784, Yixiang 1A x WGR14-636, Yixiang 1A x WGR14-642, Yixiang 1A x WGR14-646, Yixiang 1A x WGR14-657, Yixiang 1A x WGR14-668, Yixiang 1A x WGR14-766, Yixiang 1A x WGR14-774, Yixiang 1A x WGR14-785, Yixiang 1A x WGR14-792, Yixiang 1A x WGR14-798, Yixiang 1A x WGR15-428, Yixiang 1A x WGR15-443, Yixiang 1A x WGR15-447, Yixiang 1A x WGR15-459, Yixiang 1A x WGR15-466, Yixiang 1A x WGR16-776, Yixiang 1A x WGR16-786, Yixiang 1A x WGR16-789, Yixiang 1A x WGR16-792, Yixiang 1A x WGR16-804, etc. In April of the same year, the hybrid combinations together with the parent seed (sterile line has no seed, so its maintainer line is used instead) were taken to the experimental base of the State Agricultural Science Academy in Wenshan City, Yunnan Province, for planting and field restoration identification. The field test was set up with three repetitions, and then arranged randomly. The main spike of each hybrid combination was selfed in a bag,The pollen abortion (maintenance) and fertility recovery degree of the test combination are mainly investigated, and the taste quality of the test materials is also investigated; the aroma quality of the hybrid combination and its parents with self-pollination seed setting rate lower than 20.0% is identified, the leaf aroma is investigated at the waxing period, and the rest of the materials is dried after maturing, and the rice grain aroma is identified.

[0046] The test results show that there are 16 hybrid combinations with 0% self-pollination seed setting rate, 18 hybrid combinations with self-pollination seed setting rate higher than 90.0%, and the pollen abortion of the rest of the hybrid combinations is not complete, and there is pollen scattering phenomenon, the self-pollination seed setting rate is 1.0-89.0%, and most of them show semi-recovery and semi-maintenance; the aroma quality score of all the test materials is 95.5-98.2, showing a strong aroma type, and the aroma score difference between the materials is less than 5.0%, indicating that the aroma genes are controlled in an allelic manner; among the hybrid combinations showing abortion, six hybrid combinations of Wenxiang 28A×WGR14-766, Wenxiang 28A×WGR14-798, Wenxiang 28A×WGR15-466, Yixiang 1A×WGR14-766, Yixiang 1A×WGR14-798 and Yixiang 1A×WGR15-466 show complete pollen abortion (typical abortion), and especially all the green stems within 30 days after heading, indicating that the male parent not only shows good maintenance ability to cytoplasmic male sterility, but also the aroma score of the hybrid combination and its parents is higher than 97.2, belonging to a strong aroma type, and at the same time, the tillering ability is strong, the plant leaf morphology is good, and the comprehensive resistance is strong, so WGR14-766, WGR14-798 and WGR15-466 are used as the donor variety III with maintenance ability and having recessive grayish-white endosperm marker gene (g) and strong aroma gene (s) in the present application.

[0047] Example 2 Breeding of a strong aroma type soft rice maintenance line VII with recessive grayish-white endosperm marker and strong aroma and a strong aroma type soft rice three-line sterile line IX with recessive grayish-white endosperm marker and strong aroma In this embodiment, WGR14-766, WGR14-798 and WGR15-466 screened in Example 1 are selected as the donor variety III with the maintenance ability and the recessive gray-white endosperm marker trait gene (g) and the fragrance gene (s), the line Wenxiang 24B, Wenxiang 28B, Zhuangxiang 2501B, Yixiang 1B and Zhong 9B are selected as the excellent three-line maintenance line V and Wenxiang 28A is selected as the excellent three-line sterile line VIII, and the soft rice fragrance maintenance line VII with the recessive gray-white endosperm marker (gg) and the strong fragrance (ss) and the soft rice fragrance three-line sterile line IX with the recessive gray-white endosperm marker (gg) and the strong fragrance (ss) are respectively created. The specific implementation is as follows: In March 2019, in the experimental base of Wenshan State Agricultural Academy in Sanya City, Hainan Province, WGR14-766, WGR14-798 and WGR15-466 were used as female parents, and Wenxiang 24B, Wenxiang 28B, Zhuangxiang 2501B, Yixiang 1B and Zhong 9B were used as male parents to carry out double cross pairing, so as to introduce the grey-white endosperm marker gene (g) and the strong aroma gene (s) into the excellent three-line maintainer line, and obtain 15 hybrid combination seeds which aggregate the excellent genes of the grey-white endosperm marker gene (g), the maintainer gene, the strong aroma gene (s) and high combining ability. In April of the same year, the seeds of the 15 hybrid combinations and the parent materials, a total of 23 materials, were taken to the experimental base of Wenshan State Agricultural Academy in Wenshan City, Yunnan Province, for field advantage identification and taste quality and aroma quality identification. The field test was arranged in three replicates, and the random group arrangement was used, and the yield of the test combinations and the taste and aroma quality of the test materials were mainly investigated. The test results show that the yield of the hybrid combinations per mu is 648.7-714.3 kg, the amylose content of the test materials is 9.7-18.6%, the gel consistency is 48.2-94.8 mm, the alkali consumption value is 4.1-6.8, the sensory taste score is 82.4-96.8 points, the aroma score is 54.2-96.8 points, and the amplitude difference is more than 5.0%. Among them, the yield of the three hybrid combinations of WGR14-766×Wenxiang 28B, WGR14-766×Yixiang 1B and WGR15-466×Wenxiang 28B is the highest, and the yield per mu is 714.3 kg, 704.8 kg and 684.4 kg respectively; the aroma score of the three hybrid combinations and their parent materials is 94.2-96.4 points, indicating that the strong aroma gene is allelic between the three hybrid combinations and their parent materials; in particular, the amylose content of the female parent of the combination is 9.7-10.3%, the gel consistency is 89.6-94.8 mm, the alkali consumption value is 6.2-6.8, and the sensory taste score is 94.2-96.8 points, so the physical and chemical values and the sensory taste quality score are consistent, indicating that the excellent taste quality gene is allelic between the female parent materials, the hybrid parent has strong combining ability, strong tillering, yellow mature green stem and good agronomic characters. Based on the WGR14-766×Wenxiang 28B hybrid combination and its parent plant leaf morphology, it is easy to breed a maintainer line with good plant leaf morphology, and the WGR14-766×Wenxiang 28B hybrid combination is continued to be planted and selected. The grey-white endosperm marker is used as an excellent taste quality trait marker, and the grey-white endosperm marker and strong aroma are used as the main target traits for simultaneous phenotype selection. When the F4 generation is obtained by planting and selecting back and forth in the Sanya base and the Wenshan base, six stable trait lines of WGM19-324, WGM19-332, WGM19-338, WGM19-342, WGM19-347 and WGM19-350 are obtained, which have grey-white endosperm markers and strong aroma, strong tillering, yellow mature green stem and good plant leaf morphology, and are used as the aroma type soft rice maintainer line VII with the recessive grey-white endosperm marker (gg) and strong aroma (ss) in the application.

[0048] In early April 2021, in the greenhouse of the Wen Shan State Agricultural Academy in Wenshan City, Yunnan Province, Wenxiang 28A was used as the female parent and was crossed with the maintainer lines WGM19-324, WGM19-332, WGM19-338, WGM19-342, WGM19-347, and WGM19-350 to introduce male sterile cytoplasm, and six hybrid combination seeds of Wenxiang 28A x WGM19-324, Wenxiang 28A x WGM19-332, Wenxiang 28A x WGM19-338, Wenxiang 28A x WGM19-342, Wenxiang 28A x WGM19-347, and Wenxiang 28A x WGM19-350 were obtained. In May of the same year, they were planted in the greenhouse of the Wen Shan State Agricultural Academy in Wenshan City, Yunnan Province, and bagged self-pollinated at the time of ear initiation to observe the abortion of the test combinations (F1).

[0049] The results showed that the pollen abortion of the hybrid combinations Wenxiang 28A x WGM19-324, Wenxiang 28A x WGM19-332, Wenxiang 28A x WGM19-338, Wenxiang 28A x WGM19-342, Wenxiang 28A x WGM19-347, and Wenxiang 28A x WGM19-350 was complete, but the WGM19-350 maintainer line was not stable in traits and was not selected as a maintainer parent for backcrossing. Thereafter, in the greenhouse of the Wen Shan State Agricultural Academy in Wenshan City, Yunnan Province, and the base of the Wen Shan State Agricultural Academy in Wenshan City, Yunnan Province, five maintainer lines WGM19-324, WGM19-332, WGM19-338, WGM19-342, and WGM19-347 were selected as recurrent parents and were backcrossed continuously for multiple generations to BCF6, and five cytoplasmic male sterile lines WGA21-324, WGA21-332, WGA21-338, WGA21-342, and WGA21-347 were obtained, which were used as the fragrant soft rice three-line sterile line IX with the recessive gray-white endosperm marker (gg) and strong fragrance (ss) of the present application.

[0050] Example 3 Breeding of Fragrant Soft Rice Three-Line Restorer Line X with Recessive Gray-White Endosperm Marker and Strong Fragrance In the present embodiment, WGR16-786, WGR11-726 and WGR16-804 screened out in Example 1 are selected as the donor variety IV with the recovery ability and having the recessive gray endosperm marker gene (g) and the strong aroma gene (s), ten recovery lines Wenhuo 206, Wenhuo 247, Wenhuo 305, Wenhuo 324, Wenhuo 927 and Minghuo 63, Yangdao 6, Mianhuo 725, Huazhan, Yihui 1577 are selected as the excellent recovery line VI, and the three-line recovery line X of soft rice with the consistent taste quality, the control gene isogenic, the aroma gene isogenic, the recessive gray endosperm marker (gg) and the strong aroma (ss) of the three-line sterile line IX of soft rice with the aroma type is created. The specific implementation is as follows: In March 2019, in the experimental base of Wenshan State Agricultural Academy in Sanya City, Hainan Province, WGR16-786, WGR11-726 and WGR16-804 were used as female parents, and 10 restorers Wenhu 206, Wenhu 247, Wenhu 305, Wenhu 324, Wenhu 927, Minghu 63, Yangdao 6, Mianhu 725, Huazhan and Yihu 1577 were used for incomplete diallel cross. The grey-white endosperm marker trait gene (g) and the strong aroma gene (s) were introduced into the excellent restorer line, and 30 hybrid combination seeds were obtained, which aggregated the excellent trait genes of grey-white endosperm marker trait gene (g), strong aroma gene (s), high combining ability gene and strong restorer gene, i.e. WGR16-786×Wenhu 206, WGR16-786×Wenhu 247, WGR16-786×Wenhu 305, WGR16-786×Wenhu 324, WGR16-786×Wenhu 927, WGR16-786×Minghu 63, WGR16-786×Yangdao 6, WGR16-786×Mianhu 725, WGR16-786×Huazhan, WGR16-786×Yihu 1577, WGR11-726×Wenhu 206, WGR11-726×Wenhu 247, WGR11-726×Wenhu 305, WGR11-726×Wenhu 324, WGR11-726×Wenhu 927, WGR11-726×Minghu 63, WGR11-726×Yangdao 6, WGR11-726×Mianhu 725, WGR11-726×Huazhan, WGR11-726×Yihu 1577, WGR16-804×Wenhu 206, WGR16-804×Wenhu 247, WGR16-804×Wenhu 305, WGR16-804×Wenhu 324, WGR16-804×Wenhu 927, WGR16-804×Minghu 63, WGR16-804×Yangdao 6, WGR16-804×Mianhu 725, WGR16-804×Huazhan, WGR16-804×Yihu 1577. In April of the same year, the hybrid combinations together with their parents, a total of 43 materials seeds were taken to the base of Wenshan State Agricultural Academy in Wenshan City, Yunnan Province for advantage identification. Field test set three times, arranged randomly, mainly to investigate the yield and taste of the test combinations, and the quality of aroma.

[0051] The results showed that the yield of hybrid combination per mu was 656.8-884.2 kg, the amylose content of the test material was 9.7-23.4%, the gel consistency was 42.6-94.4 mm, the alkali consumption value was 3.8-6.6, the sensory taste score was 70.8-96.2 points, the aroma score was 42.0-97.2 points, and the difference was more than 5.0%. Among them, WGR16-786× Wenhu 324, WGR16-786× Huazhan, and WGR16-786× Wenhu 305 had the highest yield, with a yield of 884.2 kg, 870.7 kg, and 864.8 kg per mu, respectively. However, the amylose content of WGR16-786× Huazhan and its parent materials was 9.8-18.4%, the gel consistency was 68.2-96.6 mm, the alkali consumption value was 4.0-6.6, the sensory taste score was 72.4-96.4 points, and the aroma score was 44.1-95.7 points. The difference in physicochemical values, sensory taste quality scores, and aroma quality scores was large, and they were eliminated. The aroma scores of the remaining hybrid combinations WGR16-786× Wenhu 324 and WGR16-786× Wenhu 305 and their parent materials were 95-96.3 points, and the aroma quality scores were consistent, indicating that the three hybrid combinations and their parent materials had strong aroma genes. Especially, the amylose content of all the maternal parent materials was 9.6-10.2%, the gel consistency was 89.2-95.4 mm, the alkali consumption value was 6.1-6.6, and the sensory taste score was 94.0-96.2 points. The physicochemical values and sensory taste quality scores indicated that the maternal parent materials had excellent taste quality genes, and the hybrid parents had strong combining ability, strong tillering, yellow mature, and good agronomic traits. Since Wenhu 324 had stronger resistance, better color change after maturity, and stronger tillering than Wenhu 305, selecting WGR16-786× Wenhu 324 hybrid combination for further generation selection was easier to breed high-combining ability, multi-resistance, strong aroma, and strong tillering restorers. Therefore, WGR16-786× Wenhu 324 hybrid combination was selected for further generation planting. Subsequently, according to the restorer breeding program and standard, the gray-white endosperm marker was used as an excellent taste quality trait marker, and the gray-white endosperm marker and strong aroma were used as the main target traits for simultaneous phenotype selection. The F5 generation was planted in the bases of Sanya City, Hainan Province, and Wenshan State Agricultural Academy in Wenshan City, Yunnan Province, for two years of continuous planting and selection.In August 2021, combined with recovery plus selection, in the base of the Wen Shan Agricultural Academy in Wenshan City, Yunnan Province, using three sterile lines Wenxiang 28A and Yixiang 1A as female parents, 10 recovery lines WGR19-287, WGR19-288, WGR19-290, WGR19-297, WGR19-302, WGR19-308, WGR19-312, WGR19-316, WGR19-320 and WGR19-327 with strong comprehensive resistance, good plant type, moderate plant height, strong tillering ability, yellow-green and yellow mature, and strong aroma were selected from the hybrid offspring. 20 hybrid combination seeds were obtained by hybridization and identification. In November of the same year, 20 hybrid combination seeds were taken to Sanya City, Hainan Province, and the base of the Wen Shan Agricultural Academy for advantage identification. The identification test was set up with three repetitions, and the Yunnan hybrid rice regional test control variety Nei 6 You 5182 was used as the control. The test mainly observed the seed setting rate and yield of the hybrid combination.

[0052] The test results show that, except for the combinations Wenxiang 28A x WGR19-290, Yixiang 1A x WGR19-290, Wenxiang 28A x WGR19-327, and Yixiang 1A x WGR19-327, the seed setting rate of the rest of the combinations is more than 85.0%, indicating that the paternal recovery is good. The tillering ability of Wenxiang 28A x WGR19-287 and Yixiang 1A x WGR19-287 hybrid combinations is poor, and the yield is more than 5.0% lower than the control, indicating that the combining ability is not strong. Combined with the fact that WGR19-287 is not stable enough, it is given up. Therefore, WGR19-288, WGR19-297, WGR19-302, WGR19-308, WGR19-312, WGR19-316, and WGR19-320 are selected as the invention of the three-line restorer X of the fragrant soft rice with recessive gray-white endosperm marker (gg) and strong aroma (ss).

[0053] Example 4 Breeding of Fragrant Soft Rice Three-line Hybrid Rice Variety XI with Excellent Eating Quality, Strong Aroma, and Gray-White Anti-fake Marker In this embodiment, the five three-line sterile lines WGA21-324, WGA21-332, WGA21-338, WGA21-342 and WGA21-347 selected from the lines bred in Example 2 are used as the three-line sterile line IX of the fragrant soft rice with the recessive gray-white endosperm marker (gg) and strong fragrance (ss) of the application, and the seven restorer lines WGR19-288, WGR19-297, WGR19-302, WGR19-308, WGR19-312, WGR19-316 and WGR19-320 selected from the lines bred in Example 3 are used as the fragrant soft rice restorer line X with the recessive gray-white endosperm marker (gg) and strong fragrance (ss) of the application, and the three-line hybrid rice variety XI of fragrant soft rice with excellent eating quality, strong fragrance and gray-white anti-fake marker is bred. The specific implementation is as follows: In August 2023, in the net house of the State Agricultural Academy of Wenshan City, Yunnan Province, five sterile lines WGA21-324, WGA21-332, WGA21-338, WGA21-342 and WGA21-347 were used as the female parent, and seven restorer lines WGR19-288, WGR19-297, WGR19-302, WGR19-308, WGR19-312, WGR19-316 and WGR19-320 were used to carry out an incomplete diallel cross, and enough seeds of 35 hybrid combinations were obtained. In April 2024, with the hybrid rice variety Nei5 You5182 of Yunnan Province as the control variety, the seeds were divided into 10 parts and were respectively planted in 7 cities of Wenshan, Honghe, Dehong, Baoshan, Lincang, Xishuangbanna and Puer of Yunnan Province, and in 10 different ecological regions of Deyang City of Sichuan Province, Guiyang City of Guizhou Province and Nanning City of Guangxi Zhuang Autonomous Region for multi-point advantage identification and fragrance gene isodimorphism identification test. The test area of each plot was 0.02 mu, 360 clusters were planted in each plot, and single plants were planted without repetition. In the autumn of 2024, the plant height, ear length, effective panicles per mu, grain number per panicle, filled grain number per panicle, seed setting rate, thousand-grain weight, single plant weight, plot weight and other traits were investigated, and at the same time, the changes in the endosperm fragrance and gray-white marker color of the F1 plant rice grains were observed. Among them, the hybrid combination materials in the three test points of Wenshan City of Yunnan Province, Deyang City of Sichuan Province and Nanning City of Guangxi Zhuang Autonomous Region were planted together with the parent lines (the sterile lines were replaced by their maintainer lines), a total of 47 materials, and after drying, the seeds were milled into milled rice for sensory eating quality and fragrance quality identification.

[0054] The yield advantage identification results show that: compared with the control, the average yield of each test point is higher than that of 5you 5182, and there are 20 hybrid combinations, among which 8 combinations have a significant increase in yield, namely, WGA21-338xWGR19-288, WGA21-342xWGR19-288, WGA21-338xWGR19-316, WGA21-342xWGR19-316, WGA21-324xWGR19-288, WGA21-338xWGR19-308, WGA21-342xWGR19-308, WGA21-338 / WGR19-320; The hybrid combinations with the highest yield, strong aroma and green yellow maturity are WGA21-338xWGR19-288, WGA21-342xWGR19-288 and WGA21-338xWGR19-316, with a yield of 734.7 kg per mu, 722.4 kg per mu and 716.6 kg per mu respectively. The taste and aroma quality test results: the amylose content of hybrid combinations and their parents is 9.6-10.3%, the gel consistency is 89.4-95.8mm, the alkali consumption value is 6.2-6.8, the sensory taste score is 94.8-96.4 points, and the aroma score is 94.6-96.7 points. The results are consistent with those of Example 1, which has the ability to maintain and has the recessive gray endosperm marker gene (g) and the strong aroma gene (s). The donor variety III and the donor variety IV with restoration ability and the recessive gray endosperm marker gene (g) and the strong aroma gene (s) are consistent, and all show excellent eating quality characteristics such as pleasant aroma, soft and refreshing, rice grains are crystal and elastic and glossy, gelatinization is not sticky, and cold rice is not rehydrated, hard, and cold and hot can be eaten. Although there are slight differences in the brightness of the marker color among the test materials and in different test points, the physicochemical values, sensory eating quality scores and aroma quality scores are consistent, indicating that the excellent eating quality of all test hybrid combinations and their parent materials is consistent and the control gene is equal, the aroma quality is consistent and the gene is equal, which verifies the accuracy of the gray endosperm marker trait representing excellent eating quality, and it is completely feasible and simple and practical to use the gray endosperm marker trait to represent excellent eating quality for phenotype selection of excellent eating quality.

[0055] Among them, the sensory taste quality identification score is the highest, and the three test point score changes of the hybrid combination are the smallest, which are WGA21-338xWGR19-288, WGA21-342xWGR19-288 and WGA21-324xWGR19-288. In combination with the yield, resistance, adaptability, taste quality, aroma quality and other comprehensive performances of each test point, the hybrid combination WGA21-338xWGR19-288 has the most excellent comprehensive performance, that is, the sterile line WGA21-338 is temporarily named as the three-line sterile line Wenbai 1338A of soft rice with aromatic type, the restoration line WGR19-288 is temporarily named as the restoration line Wenbai 9288 of soft rice with aromatic type, and the hybrid combination Wenbai 1338A / Wenbai 9288 combination is temporarily named as the three-line hybrid rice variety Wenbai you 9288 of soft rice with aromatic type. Wenbai you 9288 is the three-line hybrid rice variety of soft rice with aromatic type of the present application, which has excellent taste quality and strong rice aroma, and has a gray-white anti-counterfeiting mark.

[0056] In summary, the breeding method in the present application has the following advantages: 1. The problem of detecting and identifying the taste quality and its control gene for multiple times in the selection of high-quality hybrid rice parents is solved for the first time, which ensures that the taste quality and its control gene of the hybrid parent are consistent and reduces the breeding cost.

[0057] Specifically, at present, in order to ensure that the quality of hybrid rice is consistent and the control gene is consistent, and then hybridize to form a hybrid rice variety with excellent taste quality, most of the selected single plants in the process of selecting high-quality hybrid rice parents need to be tested for taste quality and its control gene multiple times. However, due to the fact that taste quality cannot be identified by sensory phenotype, and the formation of taste quality is affected by the Wx major gene and other micro genes, as well as the protein and fat content in the endosperm, the accuracy of detection by molecular markers is limited, and it is still necessary to combine physical and chemical analysis with sensory taste quality identification, i.e. molecular marker-assisted breeding. These detection methods not only increase the workload and complexity of selecting high-quality parents, but also increase the cost of selection and investment. Taking the gray-white endosperm marker trait as a selection marker, and using soft rice with different genetic backgrounds and gray-white endosperm marker traits as high-quality soft rice genes and aroma gene donor varieties, the present application creates a soft rice maintaining line, sterile line, and restoring line with excellent taste quality and strong aroma, as well as a hybrid rice variety, without the need for detection and identification of the taste quality and its control gene of the selected parent. Because the same allele controls the gray-white endosperm trait, and there is a phenotypic trait marker, there is no need to identify the taste quality and its isogenic gene of the selected material in the selection of the maintaining line and the restoring line, i.e. as long as the maintaining material and the restoring material have the gray-white endosperm marker trait, their taste quality is excellent, and the genes controlling the taste quality of the two are isogenic. In this way, the key technical link of detecting and identifying the taste quality and its control gene of the selected maintaining line material and the restoring line material can be saved at the same time, effectively solving the key technical problem of detecting and identifying the taste quality and its control gene of the selected material multiple times due to the need to master the isogenicity of the taste quality and its control gene of the selected parent, and ensuring the consistency of the taste quality and its control gene of the selected parent of the soft rice hybrid rice with excellent taste quality and strong aroma, improving the precision of the selected parent, and reducing the cost of parent selection.

[0058] 2. For the first time, the gray-white endosperm marker gene representing excellent taste quality is used for synchronous phenotype aggregation selection with high combining ability, strong aroma, and maintaining genes, which solves the problem of difficult selection of high-quality hybrid maintaining single plants in early generations, and has strong target quality selection.

[0059] Specifically, hybrid rice eating quality is mainly affected by the quality of its sterile line, so the creation of high-compatibility, excellent eating quality, and soft rice sterile line is the main direction of hybrid rice breeding. However, the bottleneck problem is that the performance of cytoplasmic male sterility and eating quality traits cannot be identified by sensory phenotype, leading to the blindness of single plant selection and uncertainty of hybrid offspring. The reason is that most of the parent selection of hybrid offspring is single line selection, and the quality traits of early generation single plants are still separating, with transparent and consistent appearance of good and bad grains, and very little seed quantity, which cannot be used for eating quality identification and selection of single plants. Only after the traits are stable in F4 generation, the quality single plants can be selected for cytoplasmic male sterility identification to determine the quality of the backcrossing sterile line. Although molecular markers can be used to track the detection of major genes controlling eating quality formation in the selected single plants of hybrid offspring to prevent the loss of major genes controlling eating quality formation during the process of separation and gene recombination, the quality of the selected single plants cannot be accurately reflected by molecular markers, which can only be used as a reference for selection. At the same time, the use of molecular markers for tracking the detection of Wx major genes in the selected single plants will increase the cost of selection. Therefore, the gray-white endosperm marker with excellent eating quality can be used as a selection marker, and the soft rice with gray-white endosperm and strong aroma can be used as a high-quality soft rice gene and aroma gene donor variety. By introducing the gray-white endosperm marker gene, strong aroma gene, and high-compatibility gene into the excellent maintenance material, the gray-white endosperm marker gene, strong aroma gene, and high-compatibility gene can be simultaneously selected in the F1 generation of hybrid separation. After the other agronomic traits are stable, the cytoplasmic male sterility and compatibility can be identified and selected. As a result, the high-compatibility, excellent eating quality, and strong aroma soft rice sterile line can be successfully selected. This is because the gray-white endosperm and strong aroma traits are controlled by recessive genes, which can be evaluated by sensory evaluation. As long as the single plant with gray-white endosperm and strong aroma traits is selected in the F1 generation, the genotype of the two traits is pure and stable, and will not be separated and lost due to gene recombination during the selection of the next generation. Therefore, the single plant with target traits can be selected in the F1 generation for simultaneous phenotypic aggregation and directional selection, until the other agronomic traits are stable, and the cytoplasmic male sterility and compatibility are identified, so that the sterile line with high compatibility, good maintenance, gray-white endosperm, and strong aroma can be selected for backcrossing and transformation.This selection method can completely solve the key technical problem of not being able to perform high-quality directional selection due to the inability to accurately determine whether to select a single plant with excellent taste quality, can save the complexity of continuously tracking detection using molecular markers and taste identification selection after the agronomic traits are stable, and reduces the selection cost. The selection target can be accurately synchronized with the phenotype directional aggregation selection from the F1 generation, and the selection target is accurate.

[0060] 3. The gray-white endosperm soft rice marker gene is introduced into the three-line hybrid rice parent, and the three-line hybrid rice with gray-white marker and fragrant soft rice is successfully created through the identification of the main target trait aggregation response, the yield is improved, and it is beneficial to the consumer to identify the authenticity of the product and brand building.

[0061] Specifically, although the yield of high-quality hybrid rice is high, there is still a gap in the overall taste quality compared with high-quality conventional rice, especially brand high-quality rice. However, this is not a problem with hybrid rice technology itself, but rather a lack of research on the creation of high-quality rice parents with excellent taste quality, and a lack of high-quality fragrant soft rice hybrid rice parents with excellent taste quality that can be used for grouping, resulting in most brand high-quality fragrant rice having to rely on low-yield and limited-range high-quality conventional rice. With the progress of high-quality hybrid rice breeding technology and the improvement of its taste quality, the application area of high-quality fragrant soft rice hybrid rice with excellent taste quality will become more and more extensive. However, due to the fact that the appearance of various high-quality fragrant rice and non-fragrant rice, including high-quality fragrant hybrid rice, and fragrant rice packaged by brands, is almost transparent, the aroma is similar, and the sensory organs are not easy to identify, this provides an opportunity for unscrupulous imitation of pseudo-fragrant rice with synthetic flavoring agents, which damages the rights and interests of consumers and affects the healthy development of the fragrant rice market. Therefore, the gray-white endosperm marker soft rice gene with excellent taste quality and the fragrant gene are introduced into the excellent three-line hybrid rice parent, and the high-quality fragrant soft rice inbred line, sterile line and restorer line with excellent taste quality and its control gene are created, which realizes the three-line matching, and through the identification of the main target trait aggregation response, the hybrid rice not only has high yield, but also has the gray-white endosperm marker trait, which has a very obvious color visual difference compared with other sticky rice, fragrant rice and ordinary rice, which is equivalent to “marking” the excellent taste quality of the fragrant soft rice hybrid rice, making it easier for consumers to identify, which is beneficial to prevent infringement of counterfeit and substandard products, especially to improve the planting development benefit and meet the demand for excellent taste quality of fragrant soft rice.

[0062] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own emphasis. If not fully described in an individual embodiment, reference can be made to the description in other embodiments.

[0063] The above embodiments only express the implementation of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. A breeding method of a soft rice type of a three-line hybrid rice with a grayish white anti-counterfeit mark, characterized in that, Comprise the following steps: S1, respectively provide B donor varieties III with maintaining ability and with recessive gray-white endosperm marker gene and strong flavor gene, C donor varieties IV with restoring ability and with recessive gray-white endosperm marker gene and strong flavor gene; S2, select D excellent three-line maintainer lines V to carry out incomplete diallel cross with the donor varieties III, obtain hybrid seed of polymeric recessive gray-white endosperm marker gene, strong flavor gene, high combining ability gene and strong maintaining ability gene, through field advantage identification, select the hybrid combination seed with the highest yield and strong flavor to continue planting and generation, at the same time, carry out target trait synchronous phenotype polymeric selection, through multiple generations of selfing and separation selection, obtain E strong flavor soft rice three-line maintainer lines VII with recessive gray-white endosperm marker and strong flavor; select excellent three-line sterile lines VIII as female parent to cross with the strong flavor soft rice three-line maintainer lines VII, plant the hybrid test combination seed to bag selfing, take the male parent of the test combination whose cytoplasmic male sterility is complete failure or complete abortion and selfing rate is 0% as recurrent parent, carry out multiple generations of backcrossing and nuclear replacement, obtain F strong flavor soft rice three-line sterile lines IX with recessive gray-white endosperm marker and strong flavor; S3, select G excellent restorer lines VI to carry out incomplete diallel cross with the donor varieties IV, obtain hybrid seed of polymeric recessive gray-white endosperm marker gene, strong flavor gene, high combining ability gene and strong restoring ability gene, through field advantage identification, select the hybrid combination with the highest yield and strong flavor to continue planting and generation, at the same time, carry out target trait synchronous phenotype polymeric selection, through multiple generations of selfing and separation selection, obtain H strong flavor soft rice three-line restorer lines X with the same taste quality as the strong flavor soft rice three-line sterile lines IX, control gene alleles, high combining ability, strong restoring ability and recessive gray-white endosperm marker and strong flavor; S4, carry out incomplete diallel cross on the strong flavor soft rice three-line sterile lines IX and the strong flavor soft rice three-line restorer lines X, carry out target trait polymeric response identification on the obtained hybrid test combination seed, obtain strong flavor soft rice three-line hybrid rice varieties XI with gray-white anti-fake marker and strong flavor; Wherein, B, C, D, E, F, G, H are positive integers.

2. The breeding method of the soft rice type three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S1, the breeding method of the donor varieties III and IV comprises: taking excellent three-line sterile lines I as female parent, respectively cross with A strong flavor soft rice germplasm resources II with recessive gray-white endosperm marker, plant the obtained hybrid seed to bag selfing, select the male parent of the hybrid combination whose pollen is completely sterile, selfing rate is 0%, plant leaf morphology is good and strong flavor, which is the donor variety III; select the male parent of the hybrid combination whose pollen is normal, selfing rate is above 90%, plant leaf morphology is good and strong flavor, and has different genetic background from the donor variety III, which is the donor variety IV; Wherein, A is a positive integer.

3. The breeding method of the soft rice type three-line hybrid rice with the ash-white anti-counterfeit marker according to claim 2, characterized in that, The strong flavor soft rice germplasm resources II refers to rice resource varieties from home and abroad and with recessive gray-white marker trait gene and recessive strong flavor gene.

4. The breeding method of the soft rice type three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S2, the synchronous phenotypic aggregation selection of the target traits specifically comprises: in the process of generation addition, taking the recessive gray-white endosperm marker trait as the selection marker of excellent eating quality, taking the recessive gray-white endosperm marker trait and strong flavor as the main target traits, combining the husked observation and the chewing of brown rice, and performing the phenotypic aggregation line selection of the excellent eating quality and strong flavor traits from the F1 generation.

5. The breeding method of the soft rice type of the three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S2, the number of generations of the multiple generations of self-intersection and separation selection is 6-8 generations; and the number of generations of the multiple generations of backcrossing and nuclear replacement is 6-8 generations.

6. The breeding method of the soft rice type of the three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S3, the synchronous phenotypic aggregation selection of the target traits specifically comprises: in the process of generation addition, taking the recessive gray-white endosperm marker trait as the selection marker of excellent eating quality, taking the recessive gray-white endosperm marker trait and strong flavor as the main target traits, combining the husked observation and the chewing of brown rice, and performing the phenotypic aggregation line selection of the excellent eating quality and strong flavor traits from the F1 generation.

7. The breeding method of the soft rice type of the three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S3, the number of generations of the multiple generations of self-intersection and separation selection is 6-8 generations.

8. The breeding method of the soft rice type of the three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S4, the identification of the aggregation response of the target traits specifically comprises at least one of the identification of yield, adaptability, resistance, physicochemical quality, and sensory eating quality.

9. The breeding method of the soft rice type of the three-line hybrid rice with the ash-white anti-counterfeit mark according to claim 1, characterized in that, In step S4, the amylose content of the strong-flavor soft rice three-line hybrid rice variety XI is 9.6-10.3%, the gel consistency is 89.4-95.8 mm, the alkali solubility value is 6.2-6.8, the sensory eating score is 94.8-96.4, and the flavor score is 94.6-96.7; the gray-white anti-counterfeiting marker trait in the strong-flavor soft rice three-line hybrid rice variety XI is controlled by a single recessive gene, is not affected by the environment, and can be stably inherited; and the strong-flavor soft rice three-line hybrid rice variety XI comprises Wenbaiyou 9288.

10. The strong-flavor soft rice three-line hybrid rice breeding method with a gray-white anti-counterfeiting marker according to any one of claims 1-9 in the application of rice breeding.

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