A breeding method and application of a fragrant soft rice with a grayish white anti-counterfeiting mark

Abstract

The application discloses a kind of soft rice of incense type breeding method and application with grey-white anti-fake mark, belong to rice breeding technical field.In the present application, donor variety III with recessive grey-white endosperm soft rice marker gene of isogenicity and donor variety IV with strong flavor gene of isogenicity are crossed, and hybrid seed V with recessive grey-white endosperm soft rice marker gene and strong flavor trait gene is obtained, and seed with strong flavor and recessive grey-white endosperm soft rice phenotype is used as selection marker, and by selfing, characteristic screening and identification, the soft rice variety IX of incense type with grey-white anti-fake mark, strong flavor, high yield, strong resistance, wide adaptability and strong combining ability is obtained.The identification of the soft rice variety IX of incense type in the present application can not be limited by hybrid separation generation, has high precision, strong target, shortens the breeding cycle of high-quality rice, reduces cost, and improves breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of rice breeding technology, specifically relating to a method and application for breeding fragrant soft rice with a gray-white anti-counterfeiting mark. Background Technology

[0002] Rice is one of the world's most important crops, feeding over 50% of the global population and serving as the staple food for over 65% of my country's population. The aroma of rice is internationally recognized as one of the most important characteristics of rice's flavor and quality, a key indicator of high-quality rice. Internationally renowned brands of premium rice, such as Thai jasmine rice and Basmat rice from India and Pakistan, are praised worldwide for their rich aroma and pleasant taste. Li Shizhen's *Compendium of Materia Medica* records that "fragrant rice can nourish the heart and lungs, harmonize with white medicine, and prolonged consumption can lighten the body and prolong life," indicating that fragrant rice possesses functional activity and nutritional and health benefits for the human body. It can also enhance the body's anti-cancer effects, hence it is also known as functional rice. Soft rice, with its low amylose content, soft gel consistency, low gelatinization temperature, fragrant aroma, smooth and refreshing texture, non-cold hardening after cooling, edible both hot and cold, and translucent, glossy grains, has been known throughout history as "tribute rice" or "imperial rice." With the improvement of people's living standards and the enhancement of their awareness of nutrition and health in their diets, fragrant soft rice with excellent taste and health benefits is increasingly favored by consumers. To meet the growing demand for fragrant rice, my country imports large quantities of fragrant rice from countries such as Thailand and Pakistan every year; in 2017 alone, 1.12 million tons of fragrant rice were imported from Thailand. my country issued the industry standard for "Fragrant Rice" (NY / T596-2002) in 2002, and in 2008, the national standard "Sensory Evaluation Method for the Cooking Quality of Rice and Grain" (GB / T15682-2008) increased the score for strong aroma to over 40% of the sensory evaluation score. This has played a significant role in promoting the development of my country's branded fragrant rice industry and accelerating the advancement of fragrant rice breeding technology.

[0003] Due to its unique geographical and ecological conditions, as well as the lifestyle of its ethnic minorities, Yunnan is not only China's largest center of genetic and ecological diversity in rice varieties and a natural treasure trove, but also a rich center for superior rice germplasm. It preserves the world's richest and most diverse soft rice germplasm resources and is the origin of soft rice in the world's rice-growing regions. In particular, Yunnan soft rice germplasm resources, as a unique type of indica rice in Yunnan, have accumulated extremely rich and beneficial genetic resources through nearly 10,000 years of evolution and selection. Therefore, the exploration of Yunnan fragrant soft rice germplasm resources and their application in high-quality rice breeding have always attracted the attention of rice breeders both domestically and internationally. To meet consumer demand for fragrant soft rice, since the mid-1970s, universities and research institutions in Yunnan Province have fully utilized the abundant germplasm resources of Yunnan's fragrant soft rice varieties. They introduced aroma and soft rice genes from established local Yunnan soft rice varieties such as Haomuxi and Haopi into dwarf, highly resistant rice varieties like IR24, successfully breeding a number of national and ministerial-level high-quality fragrant soft rice varieties, including Dianrui 408, Diantun 502, Wendao No. 1, and Wendao No. 2. In the breeding of fragrant soft japonica rice, Japan successfully bred a number of soft rice varieties with an amylose content of around 10%, such as Milky Queen, in the 1980s through mutagenesis of the high-quality japonica rice variety Koshihikari. In 1999, the Jiangsu Academy of Agricultural Sciences and other institutions introduced the soft rice gene of the famous Japanese soft rice variety Kanto 194 into the fragrant japonica variety Wujingxiang 14, successfully breeding the high-quality fragrant soft japonica rice variety Nanjing 46, which is known as "Jiangsu's best-tasting rice." After approval and large-scale promotion, Nanjing 46 was used as a donor for soft rice and aroma genes in Jiangsu, Shanghai, Zhejiang, and other regions, successfully breeding a batch of fragrant soft japonica rice varieties with excellent eating quality and promoting their production and application. Heilongjiang Province successfully bred a batch of fragrant soft japonica rice varieties with rich aroma and excellent eating quality, adapted to cold and high-latitude regions, such as Wuyoudao 4 and Songjing 22, using high-quality Japanese japonica rice as a genetic reference, strongly supporting the brand building of "Wuchang Fragrant Rice."

[0004] However, while fragrant rice or aromatic rice is favored worldwide for its unique health and nutritional benefits, delightful aroma, and pleasant taste, resulting in strong market demand and selling prices several times, even ten times, higher than ordinary rice, and a thriving industry with good returns on investment in its cultivation and development, the market has also seen rampant counterfeiting, counterfeiting, and other illegal activities. Furthermore, unscrupulous merchants are adding illegal synthetic flavorings to ordinary rice to reap huge profits, which not only seriously infringes on intellectual property rights but also harms the physical and mental health of consumers and makes it extremely difficult for businesses to protect their rights. The main factors contributing to the problem are: Firstly, the appearance of high-quality glutinous rice on the current premium rice market is uniform, almost transparent. Secondly, genuine and counterfeit fragrant soft rice look essentially the same and have similar aromas. Although there are slight differences in anti-counterfeiting labels and illustrations on the packaging, ordinary consumers are generally not accustomed to them or cannot easily distinguish them by sensory perception. Furthermore, even if companies suspect a problem with the product's origin, verifying the authenticity requires commissioning a qualified and legal product quality inspection agency for testing and evaluation. The resulting inspection and evaluation reports often take time, making it difficult to promptly investigate and crack down on counterfeit fragrant soft rice products illegally entering the market. In particular, most ordinary consumers lack awareness of brand packaging distinctions, making them extremely vulnerable to counterfeit premium rice. By the time they discover the problem with the purchased counterfeit premium rice, losses and harm have already occurred, which consumers deeply resent. Although significant resources are invested annually in resolutely combating this counterfeiting and counterfeiting behavior, achieving some results, the overall effect remains unsatisfactory. The rampant counterfeiting in the branded soft rice market, including infringement, counterfeiting, and the illegal addition of synthetic flavorings, not only seriously harms the development of high-quality branded rice and consumers, but also hinders the creation of branded soft rice and the healthy development of the market.

[0005] Therefore, how to use biological breeding technology to "mark" the aroma of soft rice with obvious characteristics so that ordinary consumers can more easily identify it, prevent the purchase of counterfeit branded aroma of soft rice, protect the rights and interests of developers and consumers, and promote the healthy development of my country's branded aroma of soft rice industry is an important technical problem that urgently needs to be solved. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for breeding aromatic soft rice with a gray-white anti-counterfeiting mark. The aromatic soft rice obtained through the breeding method of this invention has a very obvious gray-white endosperm soft rice mark, which is easily identifiable by the naked eye and can serve as an anti-counterfeiting mark for aromatic soft rice. Furthermore, selection using this mark is not limited by the hybridization segregation generation, has high precision and strong targeting, shortens the high-quality rice breeding cycle, reduces costs, and improves breeding efficiency.

[0007] In a first aspect, the present invention provides a method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark, comprising the following steps: 1) crossing donor variety III with an allelic recessive gray-white endosperm soft rice marker gene (g) and donor variety IV with an allelic aroma-rich gene (a) to obtain a heterozygous seed V possessing both the recessive gray-white endosperm soft rice marker gene (g) and the aroma-rich trait gene (a); 2) self-pollinating the heterozygous seed V, using the aroma-rich and recessive gray-white endosperm soft rice phenotypic trait (ggaa) as selection markers to obtain seed VI (ggaa) possessing both the recessive gray-white endosperm soft rice marker trait and the aroma-rich trait; 3) self-pollinating seed VI (ggaa) for multiple generations to obtain a seed possessing both the aroma-rich and recessive gray-white endosperm soft rice phenotypic trait. Using the endosperm soft rice phenotypic trait (ggaa) as a selection marker, a soft rice lines with gray-white endosperm soft rice markings and strong aroma were obtained. These soft rice lines underwent dominance identification, quality physicochemical analysis, and taste quality assessment, resulting in b, a stable aromatic soft rice line VII (ggaa) with a latent gray-white endosperm soft rice marking and strong aroma. 4) The characteristics of aromatic soft rice line VII (ggaa) were tested, resulting in c, an aromatic soft rice variety VIII with high yield, stable traits, strong resistance, wide adaptability, gray-white endosperm anti-counterfeiting markings, and strong aroma. 5) Productivity and combining ability were assessed using aromatic soft rice variety VIII, resulting in IX, an aromatic soft rice variety with gray-white anti-counterfeiting markings, strong aroma, high yield, strong resistance, wide adaptability, and strong combining ability. Where a, b, and c are positive integers.

[0008] The hybridization in this invention is a conventional rice hybridization technique. The key points are: first, based on the biological characteristics of the male and female parents, cultivation techniques are adopted to make the parents flower at the same time; second, through external means such as artificial emasculation of the female parent (artificial emasculation is not required when the female parent is a sterile line) and artificial pollination, the pollen of the male parent is transferred to the stigma of the female parent for hybridization pollination to obtain hybrid rice seeds.

[0009] In this invention, the inventors discovered that by introducing the gene for the high-quality recessive gray-white endosperm soft rice marker into allelic fragrant rice germplasm varieties with rich flavor, the resulting fragrant soft rice varieties not only possess the quality characteristics of fragrant soft rice such as rich aroma and excellent taste, but also bear a clear marker of the high-quality recessive gray-white endosperm soft rice trait. On the one hand, consumers can easily identify genuine and counterfeit branded fragrant soft rice by simply relying on their visual senses, based on the high-quality recessive gray-white endosperm soft rice trait inherited by the fragrant soft rice itself. On the other hand, in high-quality rice breeding, using the phenotypic markers of rich aroma and high-quality gray-white endosperm soft rice as selection markers allows for precise grain and plant selection from the F1 generation onwards, without being limited by the hybridization segregation generation, through manual hulling, visual observation, and chewing of brown rice. This targeted approach can significantly shorten the breeding cycle, reduce breeding costs, and improve the efficiency of high-quality rice breeding.

[0010] Specifically, the key to this invention lies in the successful discovery of rice germplasm resources possessing the recessive high-quality gray-white endosperm soft rice marker gene (g). Based on the specific genetic characteristics of rice endosperm, and through allelic identification of the target trait gene, the independently discovered marker gene (g) for high-quality recessive gray-white endosperm soft rice and the gene for rich aroma (a) are fused together. Through gene recombination, a homozygous phenotype individual with both the rich aroma trait and the high-quality recessive gray-white endosperm soft rice marker genotype (ggaa) can be isolated from the seeds of F1 plants. Seeds with strong aroma and high-quality recessive gray-white endosperm soft rice phenotypic marker (ggaa) can be selected from F1 onwards. Grain and plant selection can be carried out by manual hulling and visual observation and chewing of brown rice. Superior single grains and plants with strong aroma and high-quality recessive gray-white endosperm soft rice phenotypic marker can be selected for self-pollination and generation selection. In this way, it is not limited by the generation of hybridization segregation, and targeted and rapid breeding can be carried out to obtain single plants and lines with stable target traits.

[0011] In some implementation schemes, in step 1), donor varieties III and IV are obtained by crossing rice germplasm resource variety I with the recessive gray-white endosperm soft rice marker trait gene (g) and aromatic rice germplasm resource variety II with the aroma trait gene (a), and then screening them after determining the allelism and combining ability of the marker gene and the aroma gene.

[0012] In some implementation schemes, rice germplasm resource variety I refers to rice germplasm resource varieties and their derivative varieties that contain the recessive gray-white endosperm soft rice marker trait gene (g) from domestic and foreign sources; and the marker trait gene refers to the endogenous gene of rice germplasm resource varieties and their derivative varieties.

[0013] In some preferred embodiments, rice germplasm resources and their derivatives include soft rice germplasm resources and their derivatives that already possess the recessive gray-white endosperm soft rice marker phenotypic trait; and rice germplasm resources and their derivatives that have not yet expressed this trait but already contain the recessive gray-white endosperm soft rice marker trait gene (g).

[0014] In some implementation schemes, rice germplasm resource variety I is obtained by hybridization of Haomulü with gray-white endosperm marker gene (g) and Yunnan soft rice variety Dianrui 408 and then screening; and Haomulü has the preservation number CCTCC NO: P202407.

[0015] In some preferred embodiments, rice germplasm resource variety I includes one or more of WR05-816, WR05-825, WR05-864, WR05-868, WR05-877, WR05-1080, WR05-882, WR05-889, WR05-890, WR05-894, WR05-896, and WR05-1007.

[0016] In some preferred embodiments, rice germplasm resource variety I includes WR05-825.

[0017] In some implementation schemes, the aromatic rice germplasm resource variety II refers to aromatic rice germplasm resource varieties and their derivative varieties that have strong aroma genes from domestic and foreign sources.

[0018] In some preferred embodiments, the aromatic rice germplasm resource variety II includes one or more of Haoxiang 24B, Wenxiang 28B, Shuixiang 285B, Haoxiang 310B, Zhuangxiang 2501B, Wenhui 184, Wenhui 206, Wenhui 242, Wenhui 247 and Wenhui 305.

[0019] In some preferred embodiments, the aromatic rice germplasm resource variety II includes Wenhui 206.

[0020] In some implementation schemes, in step 1), the recessive gray-white endosperm soft rice marker refers to the fact that after the rice grains of aromatic soft rice mature and dry, the endosperm of the brown rice and polished rice are completely opaque, showing a distinct gray-white mark, which is very easy to identify. Its amylose content is 9.2~10.8%, gel consistency ≥90mm, and alkali spreading value ≥6.0. Furthermore, the recessive gray-white endosperm soft rice marker is controlled by a single recessive gene, is not affected by the environment, and can be stably inherited.

[0021] In this invention, the aromatic soft rice with a hidden gray-white endosperm soft rice mark, after being steamed and cooked, has excellent taste and quality characteristics such as pleasant aroma, soft and refreshing taste, crystal clear and elastic grains with luster, non-sticky after gelatinization, no hardening or re-cooking after cooling, and can be eaten both hot and cold.

[0022] In some implementation schemes, in step 3), the number of generations of self-pollination in multi-generation planting is 6-8.

[0023] In some implementations, in step 4), the characteristics include at least one of high yield, adaptability, stability, resistance, genetic stability, physicochemical quality, and sensory taste quality.

[0024] In some implementation schemes, in step 5), the fragrant soft rice variety IX refers to rice whose grains are harvested and dehydrated to a moisture content of less than 13.0%. After being milled into commercial rice, it retains a rich natural rice aroma, and the whole grain of rice is grayish-white, bright and crystal clear, with a very obvious color difference. Consumers can easily distinguish the authenticity of the product by visual inspection alone. Furthermore, the fragrant soft rice variety IX is Wenbai Xianghui 726.

[0025] In a second aspect, the present invention provides the application of any of the above-mentioned methods for breeding aromatic soft rice with gray-white anti-counterfeiting marks in rice breeding.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The fragrant soft rice with gray-white endosperm and anti-counterfeiting mark obtained by the present invention has a very obvious color visual difference, which makes it very easy for consumers to distinguish when purchasing in the market, prevents the product from being counterfeited and harming health, and is conducive to intellectual property protection and brand building. 2. This invention allows for seed selection without being limited by the generation of hybridization segregation, resulting in high precision, strong targeting, reduced costs for high-quality rice breeding, and a shorter breeding cycle; 3. This invention has successfully bred a fragrant soft rice with a high-quality, latent gray-white endosperm soft rice marker trait, changing the situation of the single appearance color of high-quality rice from global brands, enriching the types of fragrant soft rice, and meeting the market's demand for diversified high-quality rice. Attached Figure Description

[0027] Figure 1 This is a flowchart of the method for breeding aromatic soft rice with gray-white anti-counterfeiting marks in this invention; Biological Preservation The *Oryza sativa* seed provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, accession number: CCTCC NO: P202407, deposit date: March 25, 2024, and classified as: *Oryza sativa* L., rice seed. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0030] Unless otherwise specified, the hybridization techniques used in the examples are conventional hybridization techniques well known to those skilled in the art.

[0031] The rice varieties involved in the following examples, including Haoxiang 24B, Wenxiang 28B, Shuixiang 285B, Haoxiang 310B, and Zhuangxiang 2501B (three-line maintainers of fragrant soft rice), Haoxiang 24A and Wenxiang 28A (three-line male-sterile lines of fragrant soft rice), and Wenhui 184, Wenhui 206, Wenhui 242, Wenhui 247, and Wenhui 305 (restorers of fragrant soft rice), are maintainers, male-sterile lines, and restorers bred by the Wenshan Academy of Agricultural Sciences. These lines are preserved in the Wenshan Academy of Agricultural Sciences germplasm bank and can also be obtained from the National Germplasm Resource Bank, which the public can access through this bank. The two-line male-sterile line Y58S was bred by the Hunan Hybrid Rice Research Center, and Long 638S and Jing 4155S were bred by the Hunan Yahua Seed Industry Research Institute, both introduced from Sichuan Longping High-Tech Seed Industry Co., Ltd. Mianyou 725 is a hybrid rice combination bred by the Mianyang Academy of Agricultural Sciences in Sichuan Province, introduced from the Mianyang Academy of Agricultural Sciences. Hongyou 6 is a high-quality, fragrant, soft-grain conventional rice variety bred by the Honghe Prefecture Academy of Agricultural Sciences in Yunnan Province. It was introduced from the Honghe Prefecture Academy of Agricultural Sciences.

[0032] Twelve rice lines, including WR05-816, WR05-825, WR05-864, WR05-868, WR05-877, WR05-1080, WR05-882, WR05-889, WR05-890, WR05-894, WR05-896, and WR05-1007, were obtained by crossing Haomulü rice, which possesses the gray-white endosperm marker gene (g), with the Yunnan soft rice variety Dianrui 408. The specific method is as follows: In September 2005, at the Rice Research Institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province, a hybridization was conducted using the Yunnan local high-quality soft rice variety Haomulü as the female parent and the Yunnan soft rice variety Dianrui 408, which carries the genetic background of Yunnan local high-quality soft rice varieties, as the male parent. In early November 2005, the obtained hybrid seeds, along with their hybrid parents, were taken to the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province, for simultaneous sowing and transplanting. The F1 hybrid combination was planted adjacent to its hybrid parents during transplanting. In early April 2006, seeds from the F1 hybrid combination Haomulü / Dianrui 408 and its hybrid parents were harvested and taken to the storage room of the Rice Research Institute of the Wenshan Academy of Agricultural Sciences for drying or sun-drying. When the seed moisture content dropped to 13%, the grains from the F1 hybrid combination Haomulü / Dianrui 408 and its hybrid parents were hulled and observed as brown rice. As a result, the endosperm of brown rice from individual plants of the Haomulü / Dianrui 408 hybrid F1 combination exhibited multiple types, including transparent, semi-transparent, partially transparent, and opaque. When compared with contrasting colors, the opaque portion was predominantly grayish-white, with a small portion showing various colors such as dark gray, milky white, cloudy, ivory, and jade. Considering that grains with grayish-white endosperm markers were relatively plump and constituted a large proportion, their traits might be more stable. Therefore, brown rice grains with grayish-white endosperm markers were selected from individual plants of the Haomulü / Dianrui 408 hybrid F1 combination and, along with opaque grains from its hybrid parent Haomulü, were subjected to physicochemical quality analyses, including amylose content, gel consistency, and gelatinization temperature. The results showed that the amylose content, gel consistency, and alkali spreading value of the gray-white endosperm marker seeds of the Haomulü / Dianrui 408 hybrid F1 combination and the opaque seeds of its hybrid maternal parent, Haomulü, were 10.2%, 90 mm, and 6.4, and 10.0%, 91 mm, and 6.2, respectively. The test results confirmed that the amylose content, gel consistency, and alkali spreading value of the gray-white endosperm marker seeds of the Haomulü / Dianrui 408 hybrid F1 combination were consistent with those of the opaque seeds of its hybrid maternal parent, Haomulü.

[0033] Therefore, in late May 2006, soft rice grains with grayish-white endosperm markings were preferentially selected from the F1 plants of the Haomulü / Dianrui 408 hybrid combination. These grains, along with the hybrid female parent Haomulü, were then self-pollinated and planted in the greenhouse of the Wenshan Academy of Agricultural Sciences in Yunnan Province until the F2 generation. In late October 2006, seeds from the F2 plants of the Haomulü / Dianrui 408 hybrid combination and the seeds from the hybrid female parent Haomulü were harvested and dried or sun-dried to 13% moisture content in the rice storage room of the Rice Research Institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province before being shelled and observed. The observation results showed that the endosperm of the F2 plant grains from the Haomulü / Dianrui 408 hybrid combination was mostly opaque, and the majority of the opaque endosperm was grayish-white. This indicates that the grains with grayish-white endosperm markings selected from the F1 plants of the Haomulü / Dianrui 408 hybrid combination, when planted for multiple generations, resulted in F2 plant seeds with the same grayish-white endosperm markings after self-pollination. In late November 2006, soft rice grains with a uniform and bright color and grayish-white endosperm marker were preferentially selected from F2 single plants of the Haomulü / Dianrui 408 hybrid combination and sown at the Wenshan Academy of Agricultural Sciences experimental base in Sanya City, Hainan Province, for self-pollination to F3. After reaching F3, at the Wenshan Academy of Agricultural Sciences experimental base in Yunnan Province and the Wenshan Academy of Agricultural Sciences experimental base in Sanya City, Hainan Province, single grains and single plants were selected based on the presence of the grayish-white soft rice marker in the endosperm. Self-pollination and selection were continuously carried out to purify the grayish-white endosperm marker soft rice gene and other superior trait genes.

[0034] By late September 2009, at the experimental base of the Wenshan Academy of Agricultural Sciences in Yunnan Province, after separation and self-pollination to F6, a relatively stable population of soft rice plants with grayish-white endosperm markers emerged. Sixteen high-yielding, resistant, and non-photosensitive plants were selected and transported to the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province in November 2009 for further propagation to F7. In March 2010, at the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province, based on factors such as photosensitivity, compact plant type, and stability... After rigorous selection and observation, four individual plants with unstable traits and weak photosensitivity were eliminated. Twelve soft rice lines with gray-white endosperm markers were successfully screened out, namely WR05-816, WR05-825, WR05-864, WR05-868, WR05-877, WR05-1080, WR05-882, WR05-889, WR05-890, WR05-894, WR05-896, and WR05-1007.

[0035] The aforementioned soft rice strains with grayish-white endosperm markers are preserved in the germplasm bank of the Wenshan Academy of Agricultural Sciences, and the public has obtained them from this germplasm bank.

[0036] The following examples refer to Figure 1 The breeding methods used are as described in the text.

[0037] Example 1: Screening of parent varieties for hybridization of high-quality recessive gray-white endosperm soft rice marker gene (g) and aroma-rich gene (a) with isoelic and high combining ability. This embodiment uses five aromatic soft rice maintainer lines with rich aroma and transparent endosperm, namely Haoxiang 24B, Wenxiang 28B, Shuixiang 285B, Haoxiang 310B, and Zhuangxiang 2501B, and five aromatic soft rice restorer lines, namely Wenhui 184, Wenhui 206, Wenhui 242, Wenhui 247, and Wenhui 305, as male parents (as aromatic rice germplasm resource variety II with the rich aroma gene (a) trait of this invention), and WR05-816, WR05-825, WR05-864, and WR05-8 Twelve soft rice lines, including WR05-877, WR05-1080, WR05-882, WR05-889, WR05-890, WR05-894, WR05-896, and WR05-1007, possessing a high-quality recessive gray-white endosperm soft rice marker gene, were used as maternal parents (as rice germplasm resource varieties I of this invention possessing the high-quality recessive gray-white endosperm soft rice marker gene (g)). Allelic donor varieties III of the high-quality recessive gray-white endosperm soft rice marker gene were screened. Details are as follows: In early October 2010, at the Wenshan Prefecture Academy of Agricultural Sciences in Yunnan Province, 12 independently bred varieties were successfully developed, including WR05-816, WR05-825, WR05-864, WR05-868, WR05-877, WR05-1080, WR05-882, WR05-889, WR05-890, WR05-894, WR05-896, and WR05-1007. Using a soft rice line with a high-quality, latent gray-white endosperm as the female parent, and five fragrant soft rice maintainer lines with transparent endosperm (Haoxiang 24B, Wenxiang 28B, Shuixiang 285B, Haoxiang 310B, Zhuangxiang 2501B) and five fragrant soft rice restorer lines (Wenhui 184, Wenhui 206, Wenhui 242, Wenhui 247, and Wenhui 305) as the male parents, pairwise hybridization was carried out to obtain 120 hybrid seed combinations.

[0038] In early November 2010, seeds of the obtained hybrid combinations were taken to the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province for viability identification. The experiment was conducted with three replicates, and the plot area was 13.5m². 2 Thirty-six clumps were transplanted into each plot, with each clump planted individually. Sowing and transplanting were carried out simultaneously. In mid-April 2011, when the experimental materials matured, a yield assessment was conducted. Seeds of the hybrid F1 combinations were harvested and taken to the rice research institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province for drying or sun-drying to remove moisture. When the seed moisture content dropped below 13%, individual seeds from each hybrid F1 combination were hulled into brown rice for observation and chewing.

[0039] The results showed that among the 120 hybrid combinations, WR05-825 / Wenhui 206, WR05-825 / Wenhui 247, and WR05-896 / Wenhui 206 had the highest yields, at 626.3 kg / mu, 602.7 kg / mu, and 589.8 kg / mu, respectively. All combinations expressed the aroma trait and the high-quality recessive gray-white endosperm marker in individual seeds. Furthermore, in most hybrid combinations, the proportion of seeds with the high-quality recessive gray-white endosperm marker and the aroma trait in individual seeds was approximately one-third, indicating that the hybrid parents of the tested rice germplasm resources, varieties I and II, contain allelic genes controlling the high-quality gray-white endosperm soft rice marker and the aroma trait. In some hybrid combinations, the proportion of aromatic seeds in individual seeds was around 50%, indicating that some lines of the tested rice germplasm resource variety I contain the allelic aroma gene. In most hybrid combinations, approximately 10.0% of the seeds from a single plant simultaneously possess both the aroma-rich trait and the high-quality recessive gray-white endosperm soft rice marker trait, with a smaller proportion reaching approximately 15.0%. From 120 hybrid combinations, the highest-yielding hybrid combination WR05-825 / Wenhui 206 was selected. Its maternal parent WR05-825 and paternal parent Wenhui 206 are the selected donor varieties of variety III (possessing the allelic, high-combining-ability gene for the high-quality recessive gray-white endosperm soft rice marker trait) and variety IV (possessing the allelic, high-combining-ability gene for the aroma-rich trait) (a).

[0040] Example 2: Breeding of a stable aromatic soft rice line VII with a rich aroma and high-quality grayish-white endosperm. In this embodiment, the hybrid progeny single plant WR05-825, which possesses the high-quality recessive gray-white endosperm soft rice marker trait selected in Example 1, was used as the donor variety III for the high-quality recessive gray-white endosperm soft rice marker trait gene (g) with allelicity and high combining ability. The Yunnan fragrant soft rice restorer line Wenhui 206, with its strong combining ability, good plant type, resistance to rice blast, high quality, and transparent endosperm, and carrying the genetic background of Yunnan local high-quality fragrant soft rice varieties, was used as the donor variety IV for the fragrant trait gene (a) with allelicity and high combining ability. A stable fragrant soft rice line VII with both fragrant aroma and high-quality gray-white endosperm soft rice marker trait was bred, specifically as follows: In August 2011, in the greenhouse of the Rice Research Institute of the Wenshan Academy of Agricultural Sciences in Wenshan City, Yunnan Province, a hybridization was conducted using the donor variety WR05-825, which possesses the allelic and highly combining gene (g) for high-quality recessive gray-white endosperm soft rice, as the female parent, and the donor variety Wenhui 206, which possesses the allelic and highly combining gene (a) for strong aroma, as the male parent. The high-quality recessive gray-white endosperm soft rice marker gene (g) was introduced into the donor variety Wenhui 206, which possesses the allelic and highly combining gene (a) for strong aroma. The high-quality recessive gray-white endosperm soft rice marker gene (g) and the strong aroma trait gene (a) were then fused to obtain a heterozygous seed V that combines the high-quality recessive gray-white endosperm soft rice marker gene (g), the strong aroma gene (a), and other superior trait genes. In early November 2011, the obtained heterozygous seed V, along with its hybrid parents, was taken to the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province for simultaneous sowing, transplanting, and self-pollination. This allowed for recombination of the target trait genes (g) and (a), as well as other superior trait genes, to form homozygous seeds with the target trait phenotype (ggaa). The F1 hybrid combination was planted adjacent to its hybrid parents during transplanting.

[0041] In early April 2012, the F1 generation of the hybrid combination WR05-825 / Wenhui 206 and its hybrid parent single-plant seeds were harvested and taken to the rice storage room of the Rice Research Institute of Wenshan Academy of Agricultural Sciences in Wenshan City, Yunnan Province for drying and dehydration. When the seed moisture content drops to 13%, the F1 plants of the hybrid combination WR05-825 / Wenhui 206 and their hybrid parent plants are hulled and chewed to observe and chew the brown rice, using the traits of strong aroma and high-quality recessive gray-white endosperm soft rice marker as selection markers. This is done to identify the expression of the target traits and select seeds (since this hybrid combination is the same as the identification combination WR05-825 / Wenhui 206 in Example 1, the hulled brown rice can be directly selected from the WR05-825 / Wenhui 206 plants in Example 1). Seeds with the genotype homozygous (ggaa) and the traits of high-quality recessive gray-white endosperm soft rice marker gene (g) and strong aroma gene (a) are selected (phenotype seed VI of this invention). Because, according to the genetic characteristics of the endosperm, the rice grains from the F1 hybrid plants are already F2 generation seeds. The genes controlling the high-quality recessive gray-white endosperm soft rice marker traits and the strong aroma trait have already segregated and recombinated, forming a homozygous ggaa genotype, resulting in seeds with both the strong aroma trait and the high-quality recessive gray-white endosperm soft rice marker phenotype. Moreover, the offspring of the recessive genes controlling the strong aroma trait and the high-quality recessive gray-white endosperm soft rice marker phenotype will not segregate and will always possess these traits.

[0042] In late April 2012, 1,200 individual plants of seeds (ggaa) that had been selected by grain selection and possessed the traits of the high-quality recessive gray-white endosperm soft rice marker gene (g) and the aroma-rich gene (a) were planted in the greenhouse of the Wenshan Academy of Agricultural Sciences in Wenshan City, Yunnan Province. By late August 2012, when the rice matured, selection was conducted using the high-quality recessive gray-white endosperm soft rice marker trait and strong aroma trait as selection markers. Specifically, seeds from 1200 individual main stem spikes were sun-dried until the seed moisture content was below 13.0%. After two months, the seeds were hulled into brown rice and observed and chewed. Based on the overall trait performance of individual plants in the field, seeds from individual plants with good overall traits such as plant type and possessing the high-quality recessive gray-white endosperm soft rice marker trait and strong aroma trait were selected and transported to the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province in late November 2012 for self-pollination, segregation, generation increase, and line selection to continuously purify the genes for the selected individual plants' superior traits. Following this method, the rice was transported between different ecological regions in Sanya and Wenshan, and self-pollinated twice a year for generation increase and line selection. By F6, a relatively stable population of fragrant soft rice plants with high-quality, latent grayish-white endosperm markers and a rich aroma emerged. From this population, 20 individual plants with compact plant type, high yield, and good resistance were selected and planted at the Wenshan Academy of Agricultural Sciences experimental base in Wenshan City, Yunnan Province in April 2016 for a comparative plant line experiment. The experiment was not replicated; the Yunnan Province high-quality conventional rice regional trial control variety Hongyou 6 was used as the control. The plot area was 13.3 m². 2 Each plot contains 360 individual plants. When mature, the individual plants are investigated for economic traits such as growth period, plant height, ear length, effective ears, number of grains per ear, seed setting rate, thousand-grain weight, and yield, as well as traits such as stability and resistance.

[0043] The results showed that 6 rice lines exhibited basically stable traits, strong resistance, and yields exceeding the control variety; 8 lines had yields close to the control variety; and 6 lines had poor resistance, unstable traits, and low yields. The rice from 14 rice lines with yields exceeding or close to the control variety, strong resistance, and stable traits underwent quality physicochemical analysis and sensory evaluation. The test results showed that 11 rice lines, including WR11-682, WR11-704, WR11-712, WR11-726, WR11-735, WR11-748, WR11-754, WR11-760, WR11-767, WR11-770, WR11-779, and WR11-784, produced rice with a rich aroma and a palatability score above 90. Simultaneously, their amylose content was above 10.0% ± 0.8%, gel consistency above 90 mm, and alkali spreading value above 6.0. Although the taste scores of three strains, WR11-718, WR11-742, and WR11-754, were also above 90, their amylose content exceeded 12.0%, their aroma was relatively mild, and their grayish-white endosperm soft rice label color was dark. Therefore, these strains were eliminated, leaving 11 strains.

[0044] Thus, in this embodiment, 11 aromatic soft rice lines with high-quality recessive gray-white endosperm soft rice marker traits and rich aroma traits, namely WR11-682, WR11-704, WR11-712, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, WR11-779, and WR11-784, were selected as the trait-stable aromatic soft rice lines VII of this invention, which are characterized by rich aroma and high-quality gray-white endosperm soft rice markers.

[0045] Example 3: Breeding of a genetically stable aromatic soft rice strain VIII with a gray-white anti-counterfeiting mark, rich aroma, excellent eating quality, and superior comprehensive agronomic traits such as high yield, adaptability, and resistance. This embodiment uses the 11 aromatic soft rice lines selected in Example 2, which possess the high-quality recessive gray-white endosperm soft rice marker trait and rich aroma trait, as the trait-stable aromatic soft rice line VII of this invention, which possesses rich aroma and high-quality gray-white endosperm soft rice marker. Genetically stable aromatic soft rice line VIII, possessing gray-white anti-counterfeiting marker, rich aroma, excellent eating quality, and excellent comprehensive agronomic traits such as high yield, adaptability, and resistance, was selected. Specifically: To accurately assess the yield, adaptability, disease resistance, and stability of the 11 aromatic soft rice lines selected in Example 2, which possess the high-quality recessive gray-white endosperm soft rice marker trait and rich aroma trait, particularly the genetic stability of the high-quality recessive gray-white endosperm soft rice marker trait, rich aroma trait, and excellent eating quality, a multi-site advantage identification experiment was conducted in April 2017 using these 11 aromatic soft rice lines as experimental materials in 10 different ecological regions: Wenshan, Jinghong, Mengzi, Luoping, Longyang, Binchuan, and Tengchong in Yunnan Province; Guiyang in Guizhou Province; Guanghan in Sichuan Province; and Nanning in Guangxi Province. The experiment was not replicated. Each plot was 0.02 mu (approximately 0.02 hectares), with 360 clumps planted per plot, planted as a single plant. In Yunnan, the control variety Hongyou 6 (a high-quality conventional rice variety from Yunnan Province) was used as the control. In Sichuan, Guizhou, and Guangxi, the main high-quality conventional rice varieties promoted locally were used as controls. At maturity, a seed evaluation survey was conducted based on plant type, tillering ability, uniformity, entire growth period, resistance to rice blast, effective panicles per mu, plant height, number of grains per panicle, seed setting rate, thousand-grain weight, and yield. Seeds from pilot projects in Wenshan, Luoping, Guiyang, and Nanning were dried and then subjected to identification of high-quality latent gray-white endosperm soft rice, aroma intensity, and excellent eating quality, as well as physicochemical analysis of quality. Based on the results of the seed evaluation surveys from each pilot project, the yields of the tested materials were all higher than the control. Among them, eight lines, including WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, and WR11-784, all showed yields more than 5.0% higher than the control. The polished rice from the pilot test materials in Wenshan, Luoping, Guiyang, and Nanning all exhibited a relatively strong rice aroma. The brightness of the high-quality recessive gray-white endosperm soft rice marker color in the polished rice from Luoping, Guiyang, and Nanning was consistent with that in Wenshan. Physicochemical analysis of the quality revealed that the amylose content, gel consistency, and alkali spreading value were within the ranges of 10.0%±0.5, 90mm±1.6, and 7.0±0.5, respectively, with an eating flavor score above 88 points. This indicates that these fragrant soft rice materials with the high-quality recessive gray-white endosperm soft rice marker and strong aroma are not sensitive to the cultivation environment and have stable genetic traits. In particular, the expression of the controlling gene for the high-quality recessive gray-white endosperm soft rice marker is not inhibited by changes in the planting environment.

[0046] In early April 2018, at the experimental base of the Wenshan Prefecture Academy of Agricultural Sciences in Sanya City, Hainan Province, eight aromatic soft rice lines (WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, and WR11-784) harvested from the Nanning pilot project were selected as male parents. These lines exhibited a high average yield (more than 5.0% higher than the control), a subtle gray-white endosperm characteristic of soft rice, and a rich aroma. These lines were then compared with the three-line system for aromatic soft rice. Eight testcross combinations were obtained by crossing the Wenxiang 28A breeding line, namely Wenxiang 28A / WR11-682, Wenxiang 28A / WR11-726, Wenxiang 28A / WR11-735, Wenxiang 28A / WR11-748, Wenxiang 28A / WR11-760, Wenxiang 28A / WR11-767, Wenxiang 28A / WR11-770, and Wenxiang 28A / WR11-784. After maturity, these seeds were harvested and planted at the Wenshan Academy of Agricultural Sciences experimental base in Wenshan City, Yunnan Province. After maturity in early September of the same year, the testcross combination seeds were harvested, dried, dehydrated, and hulled for observation. The results showed that all testcross combination seeds had fragrance, and the high-quality recessive gray-white endosperm soft rice marker trait segregated. The unmarked transparent endosperm trait was dominant over the gray-white endosperm soft rice marker trait, and the trait segregation ratio was 3:1. This confirms that the gene for strong aroma and the marker traits of grayish-white endosperm soft rice are genetically stable and will not be "lost" or not expressed due to environmental stress.

[0047] Therefore, the eight aromatic soft rice lines WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, and WR11-784, which are characterized by high yield, strong resistance, wide adaptability, and excellent quality, have a high-quality, subtle gray-white endosperm soft rice mark and a rich aroma, are identified as the genetically stable aromatic soft rice line VIII of this invention. These lines possess excellent comprehensive agronomic traits, including a gray-white anti-counterfeiting mark, rich aroma, excellent eating quality, high yield, adaptability, and resistance.

[0048] Example 4: Breeding of Wenbai Xianghui 726, a fragrant soft rice variety with gray-white anti-counterfeiting label, rich aroma, high yield, strong resistance, wide adaptability, and strong combining ability. This embodiment uses eight aromatic soft rice lines selected in Example 3—WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, and WR11-784—as the genetically stable aromatic soft rice line VIII of this invention. These lines possess excellent comprehensive agronomical traits, including a gray-white anti-counterfeiting mark, rich aroma, superior eating quality, high yield, adaptability, and resistance. An aromatic soft rice variety IX with a gray-white anti-counterfeiting mark, rich aroma, high yield, strong resistance, wide adaptability, and strong combining ability was also selected. Details are as follows: In early April 2019, eight aromatic soft rice lines—WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, and WR11-784—were successfully bred as the test materials for this invention. These lines are genetically stable and possess excellent comprehensive agronomic traits such as a gray-white anti-counterfeiting mark, rich aroma, superior eating quality, high yield, adaptability, and resistance. The Yunnan Province conventional high-quality rice regional trial control variety, Hongyou 6, was used as the control. A variety comparison trial and demonstration were conducted. The trial was held at the Wenshan Prefecture Academy of Agricultural Sciences experimental base in Yunnan Province. Each plot was 0.02 mu (approximately 0.02 hectares), with 400 clumps planted per plot, single-planted, and replicated three times. When the experimental materials matured in September 2019, a survey was conducted to evaluate the varieties based on the following parameters: effective panicles per mu, plant height, panicle length, number of grains per panicle, number of filled grains per panicle, seed setting rate, thousand-grain weight, single plant weight, and plot weight. The experimental results showed that the yields of two lines, WR11-726 and WR11-748, were more than 5.2% higher than the control variety Hongyou 6, while the yield increases of the other lines were not significant. The highest yield was achieved by line WR11-726, reaching 602.8 kg / mu, a 5.8% increase over the control variety Hongyou 6, and it also exhibited the best yield characteristics and the strongest resistance. High-yield cultivation technology demonstration sites were set up in ecological rice-growing areas at different altitudes in Wenshan, Yanshan, and Funing counties of Wenshan Prefecture and Menghai County of Xishuangbanna Prefecture. Hongyou 6, a fragrant soft rice variety widely promoted in Yunnan Province at the time, was used as the control variety. Each line was planted at 1.0 mu at each demonstration site, and management was carried out according to the local rice planting and management standards of each demonstration site. As a result, three varieties—WR11-726, WR11-748, and WR11-784—achieved an average yield per mu (667 square meters) that was more than 5.0% higher than the control at all demonstration sites. Their yields were 608.5 kg, 594.3 kg, and 587.6 kg per mu, respectively. Variety WR11-726 exhibited the highest yield, strongest resistance to rice blast, and best color change after maturity at all demonstration sites.

[0049] Meanwhile, to accurately assess the combining ability of the successfully bred aromatic soft rice line VIII, which possesses a gray-white anti-counterfeiting mark and a rich aroma, in July 2019, at the experimental base of the Wenshan Academy of Agricultural Sciences in Wenshan City, Yunnan Province, pairwise hybridization was conducted using the three-line sterile lines with good transparency (Haoxiang 24A, Wenxiang 28A, Zhuangxiang 2051A) and the two-line sterile lines (Y58S, Long 638S, Jing 4155S) as female parents, and the eight aromatic soft rice lines with gray-white anti-counterfeiting marks and rich aroma (WR11-682, WR11-726, WR11-735, WR11-748, WR11-760, WR11-767, WR11-770, WR11-784) selected in Example 3 as male parents. This yielded 48 test-cross combinations. These were planted at the experimental base of the Wenshan Academy of Agricultural Sciences in Wenshan City, Yunnan Province in April 2020. The experiment was conducted with three replicates, with each plot measuring 0.02 mu (approximately 0.02 hectares). 360 clumps were planted per plot, using Mianyou 725, a high-quality hybrid rice variety from Yunnan Province, as the control. In September 2020, when the tested hybrids matured, a survey was conducted based on factors such as effective panicles per mu, plant height, panicle length, number of grains per panicle, number of filled grains per panicle, seed setting rate, thousand-grain weight, single-plant weight, and plot yield.

[0050] The experimental results showed that eight tested combinations, including Wenxiang 28A / WR11-726, Jing 4155S / WR11-726, Haoxiang 24A / WR11-726, Zhuangxiang 2051A / WR11-726, Y58S / WR11-726, Wenxiang 28A / WR11-784, Haoxiang 24A / WR11-784, and Jing 4155S / WR11-682, had higher yields than the control. Among them, Wenxiang 28A / WR11-726, Jing 4155S / WR11-726, Haoxiang 24A / WR11-726, and Zhuangxiang 2051A / WR11-726 had yields that increased by more than 5.0% compared to the control. The Wenxiang 28A / WR11-726 combination had the highest yield, reaching 684.8 kg / mu. The experimental results show that the WR11-726 variety not only has the highest yield as a conventional rice, but also, when used as the male parent and tested with multiple sterile lines, most of the tested combinations had higher yields than the control, demonstrating the strongest combining ability. It has good application prospects as a restorer line.

[0051] Since the WR11-726 strain exhibits superior traits such as rich aroma, optimal quality, highest yield, strongest combining ability, strong resistance, and high yield in both quality physicochemical analysis and taste quality assessment, as well as in comparative tests and combined combination advantage assessment tests, especially with stable genetic inheritance of the high-quality recessive gray-white endosperm soft rice marker trait, WR11-726 is tentatively named Wenbai Xianghui 726, which is the fragrant soft rice variety IX of this invention with gray-white anti-counterfeiting marker, rich aroma, high yield, strong resistance, wide adaptability, and strong combining ability.

[0052] In summary, the breeding method of this invention has the following advantages: 1. The fragrant soft rice with gray-white endosperm and anti-counterfeiting mark obtained by the present invention has a very obvious color visual difference, which makes it very easy for consumers to distinguish when purchasing in the market, prevents the product from being counterfeited and harming health, and is conducive to intellectual property protection and brand building. Specifically, equating rice endosperm transparency with high-quality rice is merely a consumer habit, or a psychological expectation among consumers when judging rice quality and taste. The key lies in the rice's inherent flavor, nutritional and health benefits, and cooking quality. Highly transparent rice does not necessarily possess superior flavor, nutritional and health benefits, or cooking quality. However, equating endosperm transparency with quality and making it a primary goal in high-quality rice breeding has several drawbacks. From a genetic breeding perspective, this leads to increasingly homogenized high-quality rice varieties, making high-quality breeding more difficult. From a market perspective, it results in a uniform appearance of rice, with genuine and counterfeit rice, regardless of quality, making it difficult for consumers, developers, and government oversight departments to distinguish between genuine and counterfeit high-quality rice. This is particularly problematic for branded premium rice, leading to persistent counterfeiting and deceptive practices, especially the illegal addition of synthetic flavorings to famous fragrant rice brands for profit. This not only severely infringes on intellectual property rights but also harms consumer health and complicates the work of government enforcement and businesses in protecting their rights. This invention introduces a gene, independently discovered as a marker gene for the superior recessive grayish-white endosperm soft rice phenotype, into a fragrant rice germplasm variety with rich aroma and excellent eating quality. This successfully breeds a fragrant soft rice variety whose brown and polished rice exhibit very obvious and easily identifiable superior recessive grayish-white endosperm soft rice phenotype, as well as rich aroma and excellent eating quality. After being milled and marketed, this variety not only retains a rich natural rice aroma but also has a grayish-white grain. Bright and translucent, this rice exhibits a very distinct color difference compared to other common glutinous rice, especially fragrant rice. This allows consumers, government law enforcement agencies, and developers to easily distinguish genuine from counterfeit high-quality rice products on-site, without the need for instruments or equipment, relying solely on their naked senses. This is beneficial for preventing infringement and counterfeiting of domestically branded high-quality rice in the market, protecting the rights and interests of developers and consumers, and facilitating the rights protection work of government market supervision departments and enterprises. It is of great significance for building fragrant soft rice brands in my country and promoting the healthy development of the high-quality rice market.

[0053] 2. This invention allows for seed selection without being limited by the generation of hybridization segregation, resulting in high precision, strong targeting, reduced costs for high-quality rice breeding, and a shorter breeding cycle; Specifically, the genetic makeup of rice endosperm is complex, and most traits related to high-quality eating taste are controlled by recessive genes. Except for aroma traits, it is generally difficult to accurately determine the eating quality of selected individual grains or plants in early hybrid generations using human senses alone. Expensive laboratory equipment is often required for testing and comprehensive analysis. However, sometimes quality predictions based solely on laboratory data do not match actual sensory evaluation results, because rice with high-quality physicochemical indicators does not necessarily have good eating taste. Furthermore, physicochemical analysis of selected individual grains in early generations may require pulverizing the seed endosperm, and damaged seeds are often difficult to cultivate for subsequent generations. Therefore, most rice breeders generally only assess the eating quality of selected individual plants in mid-to-high generations or after quality traits have stabilized. This not only leads to a lack of direction in high-quality rice breeding, especially for high-quality aromatic soft rice, and complicated breeding procedures, but also presents technical challenges such as extended breeding cycles and increased investment. With the increasing maturity of molecular marker-assisted breeding and transgenic breeding technologies, it is possible to conduct targeted selection of genotypes for superior traits such as soft rice and aroma in the segregating generations of hybrids, regardless of environmental conditions. This has accelerated the breeding process and achieved significant results. However, molecular marker-assisted breeding also faces challenges such as high testing costs and insufficient accuracy. In the past, rice breeders proposed using the appearance of opaque or semi-transparent endosperm for phenotypic selection, selecting single grains and plants with transparent endosperm, and eliminating opaque or semi-transparent single plants as early as possible to reduce the selection population and improve selection efficiency. However, because the transparent endosperm trait is dominant over the non-transparent endosperm trait, selecting single grains and plants with transparent endosperm will still result in the segregation of opaque and transparent endosperm single plants. Targeted selection cannot be carried out in the early segregating generations of hybrids, and the problems of blind breeding and long cycles have not yet been overcome.

[0054] This invention successfully discovered rice germplasm resources with a superior recessive gray-white endosperm soft rice marker trait from high-quality soft rice germplasm resources in Yunnan. By employing hybridization and other methods, the gene for this marker trait was introduced into aromatic rice germplasm varieties. The alleles controlling the superior recessive gray-white endosperm soft rice marker trait, the aroma trait, and other superior traits were fused together. Through allele recombination, homozygous individuals expressing the superior recessive gray-white endosperm soft rice marker trait and the aroma trait were formed using the ggaa genotype. Seeds exhibiting both the superior recessive gray-white endosperm soft rice marker trait and the aroma trait (actually already F2 generation seeds) could be isolated from the F1 generation seeds. Further generation yielded hybrid offspring that were all single grains and single plants exhibiting both the superior recessive gray-white endosperm soft rice marker trait and the aroma trait. Therefore, by combining the aroma-rich trait controlled by recessive genes with the high-quality recessive gray-white endosperm soft rice phenotypic trait, and by observing the presence of the high-quality recessive gray-white endosperm soft rice marker trait and aroma-rich trait when chewing brown rice after hulling, it is possible to directly select single grains and single plants with aroma-rich trait and high-quality recessive gray-white endosperm soft rice marker trait from the F1 generation onwards, without being affected by the hybrid segregation generation.

[0055] Simultaneously, combining the marker gene for the superior recessive gray-white endosperm soft rice with the aroma gene, and using the simultaneous presence of the superior recessive gray-white endosperm soft rice phenotypic trait and the aroma-rich trait as selection markers, is not only a pioneering approach internationally, but also allows for precise selection of superior single grains and plants from F1 onwards through each generation of hybridization without the need for specialized instruments and equipment. Essentially, from F1 onwards, by using the simultaneous presence of the superior recessive gray-white endosperm soft rice phenotypic trait and the aroma-rich trait as selection markers, the selected single grains and plants are all aromatic soft rice materials with excellent eating quality, demonstrating strong target-oriented selection. Compared to currently reported methods for high-quality rice breeding, especially aromatic soft rice breeding, this method is not only accurate, rapid, simple, and practical, but also highly targeted, allowing for precise selection of superior grains and plants from the F1 segregating generation, saving manpower and resources, shortening the breeding cycle, and improving breeding efficiency.

[0056] 3. This invention has successfully bred a fragrant soft rice with a high-quality latent gray-white endosperm soft rice marker trait, which has changed the situation of the single appearance color of high-quality rice from global brands, enriched the types of fragrant soft rice, and met the market's demand for diversified high-quality rice. Specifically, high-quality fragrant rice, with its pleasant aroma and delightful taste, has won worldwide favor. Its cultivation has a long history and wide distribution globally. Through nearly ten thousand years of natural and human-induced selection, it has developed germplasm resources adapted to cultivation in various parts of the world. Therefore, the global fragrant rice germplasm resources are abundant and diverse. my country is one of the world's centers of origin for cultivated rice, possessing exceptionally rich fragrant rice germplasm resources. Fragrant rice is traditionally known as "tribute rice," with Yunnan fragrant soft rice being the most famous. As early as the mid-1970s, research institutes and universities in Yunnan used local high-quality soft rice varieties as donor varieties for high-quality soft rice genes, hybridizing them with improved rice varieties. This successfully bred a number of national gold medal-winning high-quality fragrant soft rice varieties, such as Dianrui 408, Wendao No. 1, and No. 2, which have been widely promoted and applied in production. However, possibly due to the influence of chalky white inferiority, people used to equate good transparency with high quality. Therefore, most of the high-quality rice varieties at home and abroad are of the type with high transparency. Breeders also follow the market trend and regard high transparency as an important goal of high-quality breeding. This has led to a shortage of high-quality rice germplasm resources. In addition, the requirement to meet the transparency standard in breeding goals has made the high-quality rice varieties increasingly homogeneous. In particular, the appearance of high-quality fragrant soft rice brands is basically transparent, which narrows the choice for consumers. In addition, the aroma is not easy to identify, which is also an important factor that makes it difficult to stop the counterfeiting, imitation and even illegal addition of flavorings to branded fragrant soft rice.

[0057] Studies on the relationship between rice starch tissue structure and eating quality have demonstrated that transparency and eating quality are not positively correlated; many varieties with high transparency and high amylose content are not necessarily delicious. Based on these factors, the applicant of this invention has successfully introduced a recessive gray-white endosperm soft rice marker gene, which they independently discovered, into a fragrant rice germplasm variety with excellent eating quality through hybridization. Using the traits of strong aroma and high-quality recessive gray-white endosperm soft rice as selection markers, they have successfully bred the fragrant soft rice variety Wenbaixianghui 726, which possesses a strong aroma and a high-quality recessive gray-white endosperm soft rice marker. This fills the international gap of lacking a high-quality fragrant soft rice variety with a strong aroma and a high-quality recessive gray-white endosperm soft rice marker. The promotion and development of these high-quality fragrant soft rice varieties with a strong aroma and a high-quality gray-white endosperm soft rice marker will play a significant role in changing the monotonous appearance and color of high-quality branded rice in my country, enriching the types of high-quality branded rice, and meeting the increasing demand for higher and more diversified rice quality as people's living standards improve.

[0058] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0059] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for breeding aromatic soft rice with a gray-white anti-counterfeiting label, characterized in that, Includes the following steps: 1) Cross the donor variety III, which has an allelic recessive gray-white endosperm soft rice marker gene, with the donor variety IV, which has an allelic aroma-rich gene, to obtain a heterozygous seed V that simultaneously possesses the recessive gray-white endosperm soft rice marker gene and the aroma-rich trait gene. 2) The heterozygous seed V was planted and self-pollinated, and the seeds with strong aroma and recessive gray-white endosperm soft rice phenotype were used as selection markers to obtain seed VI with recessive gray-white endosperm soft rice marker phenotype and strong aroma phenotype. 3) Seed VI was self-pollinated for multiple generations. Using the seeds’ strong aroma and recessive gray-white endosperm soft rice phenotypic traits as selection markers, a soft rice line with gray-white endosperm soft rice marker and strong aroma was obtained. The soft rice line was subjected to dominance identification, quality physicochemical analysis and taste quality identification to obtain b stable aroma soft rice line VII with recessive gray-white endosperm soft rice marker and strong aroma. 4) The characteristics of the aromatic soft rice strain VII were tested, and c aromatic soft rice strain VIII with high yield, stable traits, strong resistance, wide adaptability, gray-white anti-counterfeiting mark in endosperm and rich aroma were obtained. 5) The productivity and combining ability of the aforementioned aromatic soft rice strain VIII were evaluated to obtain aromatic soft rice strain IX, which has a gray-white anti-counterfeiting mark, rich aroma, high yield, strong resistance, wide adaptability and strong combining ability; Where a, b, and c are positive integers; In step 1), the donor varieties III and IV are obtained by crossing rice germplasm resource variety I, which has a recessive gray-white endosperm soft rice marker trait gene, and aromatic rice germplasm resource variety II, which has a strong aroma trait gene, and then screening them after determining the allelism and combining ability of the marker gene and the aroma gene. The rice germplasm resource variety I was obtained by hybridization of Haomulü, which has a gray-white endosperm marker gene, and Yunnan soft rice variety Dianrui 408, and the preservation number of Haomulü is CCTCC NO: P202407.

2. The method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark according to claim 1, characterized in that, The aromatic rice germplasm resource variety II refers to aromatic rice germplasm resource varieties and their derivative varieties that have strong aroma genes from both domestic and foreign sources.

3. The method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark according to claim 1, characterized in that, In step 1), the recessive gray-white endosperm soft rice marker refers to the fact that after the rice grains of aromatic soft rice mature and dry, the endosperm of the brown rice and polished rice are completely opaque, showing a distinct gray-white mark, which is very easy to identify. Its amylose content is 9.2~10.8%, gel consistency ≥90mm, and alkali spreading value ≥6.

0. Furthermore, the recessive gray-white endosperm soft rice marker is controlled by a single recessive gene, is not affected by the environment, and can be stably inherited.

4. The method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark according to claim 1, characterized in that, In step 3), the number of generations of self-pollination in the multi-generation planting is 6-8 generations.

5. The method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark according to claim 1, characterized in that, In step 4), the characteristics include at least one of high yield, adaptability, stability, resistance, genetic stability, physicochemical quality and sensory taste quality.

6. The method for breeding aromatic soft rice with a gray-white anti-counterfeiting mark according to claim 1, characterized in that, In step 5), the fragrant soft rice variety IX refers to rice whose grains are harvested and dehydrated to a moisture content of less than 13.0%. After being milled and processed into commercial rice, it retains a rich natural rice aroma. Moreover, the whole grain of rice is grayish-white, bright and crystal clear, with a very obvious color difference. Consumers can easily distinguish the authenticity of the product by visual inspection alone. Furthermore, the fragrant soft rice variety IX is Wenbai Xianghui 726.

7. The application of the rice breeding method with gray-white anti-counterfeiting mark as described in any one of claims 1-6 in rice breeding.

Images