Breeding method and application of soft rice with recessive albino endosperm marker

Abstract

The application discloses a soft rice rice breeding method with recessive gray-white endosperm marker and application, and belongs to the technical field of rice breeding. The method comprises the following steps: firstly, a soft rice donor variety III with a gray-white endosperm marker gene is screened; then, through hybridization and taking the gray-white endosperm as a breeding marker, a soft rice strain VI with the gray-white endosperm marker is obtained after multiple self-crossing generations; then, the characteristics of the soft rice strain VI and the genetic traits of the gray-white endosperm marker are tested, and a soft rice strain VII with the recessive gray-white endosperm marker controlled by a recessive single gene is obtained; finally, yield testing is carried out on the soft rice strain VII, and the soft rice IX with the recessive gray-white endosperm marker is obtained through the identification of combining ability after hybridization with a sterile fine variety VIII. The method is not limited by the hybridization separation generation, has high precision, high target, reduces the cost of high-quality rice breeding, and shortens the breeding period.

Description

Technical Field

[0001] This invention belongs to the field of rice breeding technology, specifically relating to a breeding method and application of soft rice with a latent gray-white endosperm marker. Background Technology

[0002] Rice is one of the world's most important crops, feeding over 50% of the global population, and in my country, over 65% of the population relies on rice as their staple food. Soft rice, with its low amylose content, soft gel consistency, low gelatinization temperature, fragrant aroma, smooth and refreshing texture, non-cold hardening after cooling, edibility both hot and cold, and glossy grains, has long been known as "tribute rice" and "imperial rice," enjoying immense popularity among consumers. It is also a high-end raw material for making rice-based snacks, instant rice, and other fast-food products, with a broad market demand. Yunnan, due to its unique geographical and ecological conditions and the lifestyle of its ethnic minorities, is not only China's largest center of rice genetic and ecological diversity and a natural treasure trove, but also a rich center for superior rice germplasm, preserving the world's richest and most diverse soft rice germplasm resources, making it the world's authentic origin of soft rice. In particular, Yunnan soft rice germplasm resources, as a unique type of indica rice in Yunnan, have accumulated a wealth of "favorable" genetic resources through thousands of years of evolution and selection. Therefore, the exploration of Yunnan soft rice germplasm resources and their application in high-quality rice breeding have always attracted much attention from the rice breeding scientific community at home and abroad.

[0003] Although Yunnan's local soft rice varieties have excellent eating quality, their grain transparency is poor. The endosperm of most grains exhibits a mixture of transparent, semi-transparent, and partially transparent characteristics, with opaque endosperm often appearing dark white, resulting in poor appearance. Furthermore, their tall stalks lead to poor resistance and a narrow range of adaptability. Therefore, since the mid-1970s, universities and research institutions in Yunnan have introduced the soft rice genes from established local 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 rice varieties, including Dianrui 408, Diantun 502, Wendao No. 1, and Wendao No. 2. However, the eating quality of these varieties still cannot match that of established local soft rice varieties like Haomuxi and Haopi, limiting their widespread adoption.

[0004] However, the genetic characteristics of rice endosperm, which is consumed by humans, are more complex than other traits, making the improvement of high-quality rice endosperm more difficult. First, regardless of whether it's indica or japonica rice, endosperm transparency deteriorates as the amylose content decreases. Currently, there are no reports of breeding rice varieties with an amylose content of around 10% and endosperm transparency meeting national standards. Although some scholars have attempted to solve the problem of soft rice transparency through the discovery of new germplasm resources and gene editing, there are still no reports of successful breeding. Some breeders have increased the amylose content to meet transparency requirements, often leading to a decline in the eating quality of the bred varieties. Therefore, achieving both high-quality soft rice and good transparency remains a major technical challenge in breeding high-quality soft rice. Second, from the perspective of field selection, because endosperm transparency is dominant over opacity, selecting individual plants with high endosperm transparency may also result in materials with medium to high amylose content. Materials with good endosperm transparency are not necessarily high-quality soft rice plants. Furthermore, even hybrid offspring with good transparency can separate into transparent and opaque individual plants. Therefore, it is difficult to predict the quality of selected plants through sensory evaluation during field selection. Laboratory equipment is essential for comprehensive analysis using molecular markers, starch tissue structure analysis, and physicochemical analysis. Otherwise, it is difficult to accurately select high-quality individual grains and plants, leading to a longer breeding cycle and higher costs for high-quality rice. Thirdly, the eating quality of rice endosperm is not only related to its amylose content but also to its gel consistency, gelation temperature, and other properties, and is also affected by its amylopectin structure. Because different varieties with similar amylose content, gel consistency, and gelation temperature often exhibit significant differences in eating quality, it remains difficult to precisely improve the eating quality of rice endosperm by adjusting endosperm amylose content, gel consistency, and gelation temperature in current rice breeding.

[0005] In summary, the existing breeding processes for soft rice have problems such as long breeding cycles, low efficiency, and poor eating quality. Summary of the Invention

[0006] The purpose of this invention is to provide a breeding method and application for soft-rice varieties with latent gray-white endosperm markers. This addresses the problems of long breeding cycles, low efficiency, and poor eating quality in existing soft-rice breeding processes.

[0007] In a first aspect, the present invention provides a method for breeding soft rice with a recessive gray-white endosperm marker, characterized by comprising the following steps: 1) hybridizing a soft rice germplasm resource variety I with endosperm color-specific and a rice variety II with transparent endosperm, and after screening, obtaining a soft rice donor variety III with a gray-white endosperm marker gene; 2) hybridizing soft rice donor variety III with a soft rice variety IV to obtain hybrid seed V, using gray-white endosperm as a breeding marker, and after multiple self-pollination generations of the F1 generation of hybrid seed V, obtaining b soft rice lines VI with a gray-white endosperm marker; 3) testing the characteristics of soft rice line VI and the genetic traits of the gray-white endosperm marker, obtaining c soft rice lines VII with a recessive gray-white endosperm marker controlled by a recessive single gene; 4) conducting yield tests on soft rice line VII, and simultaneously hybridizing it with a sterile line VIII and then performing combining ability identification, obtaining soft rice IX with a recessive gray-white endosperm marker; wherein, a, b, and c are positive integers.

[0008] In this invention, given the genetic characteristics of rice endosperm and the fact that the color phenotypic trait of rice endosperm for human consumption directly reflects the internal structure and composition of the endosperm itself, i.e., its quality, and that the colored endosperm trait is recessive to the transparent endosperm trait, successfully identifying the colored endosperm trait as a selection marker for high-quality soft rice would benefit breeders by allowing them to visually identify brown rice from the F1 segregating generation onwards, directly observing the hulled hybrid offspring. This would enable rapid and accurate selection of single grains and individual plants of hybrid offspring with high-quality colored endosperm markers. Yunnan's local high-quality soft rice germplasm resources constitute a mixed population, with rice endosperm being mostly a mixture of translucent, partially transparent, and opaque, indicating rich genetic diversity in its colored endosperm and containing abundant genes for opaque endosperm. Therefore, identifying and screening for recessive colored endosperm as a selection marker for high-quality soft rice is the most feasible and relatively easy approach.

[0009] Furthermore, this invention first screens a donor variety III with a gray-white endosperm marker gene from numerous Yunnan local high-quality soft rice germplasm resources, variety I (which includes varieties with translucent, partially transparent, and opaque seeds and grains, and diverse endosperm colors). This gray-white endosperm marker gene is stably inherited and is correlated with or even linked to high-quality eating traits such as low amylose content, high gel consistency, and low gelatinization temperature. Then, this donor variety III is crossed with a high-quality soft rice variety IV with a genetic background of Yunnan local high-quality soft rice germplasm resources. Using the high-quality gray-white endosperm trait as a breeding marker, starting from F1... Each hybrid segregating generation directly screens for single grains and plants exhibiting the marker trait by visual inspection after hulling. Through multiple generations of self-pollination, a soft rice line VI with a high-quality grayish-white endosperm marker is obtained. Then, this soft rice line VI undergoes multi-location field trials, quality assessment, and marker trait genetic analysis to obtain a high-quality, high-yielding, and multi-resistant soft rice variety VII with a high-quality recessive grayish-white endosperm marker. Finally, productivity and combining ability are assessed using soft rice variety VII to obtain a high-quality soft rice variety IX with excellent quality, strong combining ability, high yield, wide adaptability, bright and uniform grayish-white endosperm color, obvious marker color, and controlled by a recessive single gene. This invention's soft rice breeding method is not limited by hybrid segregating generations, has high precision and strong targeting, reduces the cost of high-quality rice breeding, shortens the breeding cycle, and improves the efficiency of high-quality rice breeding.

[0010] In this invention, hybridization is a conventional rice hybridization technique. The key points include: 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.

[0011] In some implementations, soft rice with a recessive gray-white endosperm marker refers to soft rice whose brown rice endosperm is gray-white, has an amylose content of 9.5-10.5%, a gel consistency of ≥90mm, and an alkali spreading value of ≥6.0 after the rice grains have matured and dried (moisture content ≤13%); and the recessive gray-white endosperm marker is controlled by a single recessive gene and can be stably inherited.

[0012] In some implementation schemes, in step 1), endosperm color specificity refers to the different endosperm colors among different grains in the same ear and among different individual plants; endosperm color specifically includes: transparent and / or opaque; opaque colors include one or more of dark gray, milky white, cloudy, grayish white, silvery white, ivory, mutton fat, and jade, and the endosperm color specificity varies greatly among different soft rice germplasm resource varieties I.

[0013] In some implementation schemes, in step 1), the soft rice germplasm resource variety I is derived from local soft rice germplasm resources and their derivative varieties from Yunnan.

[0014] In some preferred embodiments, Yunnan local soft rice germplasm resources and their derivative varieties include one or more of the following: Haomulü, Haomuxi, Haopi, Haomuyun, Babaogu, Haomuya, Haobayuan, Haojiehai, Haodilahao, Haoerwanghanduo, Haoanmen, Haoannong, Haogongxin, Haodadanong, Haogan, Haoha, Haoleng, Haokang, Haomuliang, and Haozhao.

[0015] In some implementation schemes, in step 1), the rice variety II with transparent endosperm is a rice variety with endosperm transparency reaching or exceeding the national standard level 3, and which does not contain photosensitive genes, has a short growth period, good high-yield traits, and strong resistance.

[0016] In some preferred embodiments, the rice variety II with transparent endosperm includes one or more of Haoxiang 24B, Wenxiang 28B, Wenhui 184, Wenhui 206, and Wenhui 247.

[0017] In some implementations, in step 1), the soft rice donor variety III with the gray-white endosperm marker gene is Haomulü, and the Haomulü preservation number is CCTCC NO: P202407.

[0018] In some implementation schemes, in step 2), the soft rice superior variety IV is obtained by hybridization and selection using soft rice germplasm resource variety I with endosperm color as the parent; the number of generations of self-pollination is 6-8.

[0019] In some preferred embodiments, the soft rice variety IV is Dianrui 408.

[0020] In some implementations, in step 3), the characteristics of the soft rice line VI include yield, resistance, adaptability, and stability.

[0021] In some implementations, in step 4), the soft rice IX with a latent gray-white endosperm marker is Wenbaihui 825.

[0022] In a second aspect, the present invention provides the application of any of the above-mentioned methods for breeding soft rice with recessive gray-white endosperm markers in rice breeding.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The seed selection of this invention is not limited by the hybridization segregation generation, and has high precision and strong targeting, which reduces the cost of high-quality rice breeding and shortens the breeding cycle; 2. This invention is the first to systematically discover high-quality soft rice germplasm resources with high-quality recessive gray-white endosperm markers from high-quality soft rice germplasm resources in Yunnan, which enriches the types of high-quality rice and meets the market's diversified demand for high-quality rice. 3. This invention utilizes high-quality local soft rice germplasm resources in Yunnan to successfully discover high-quality soft rice gene resources with high-quality recessive gray-white endosperm markers. The discovery and utilization of materials are from a wide range of sources, the discovery method is simple, and the cost is low. Attached Figure Description

[0024] Figure 1 This is a flowchart of the breeding method for soft rice with latent gray-white endosperm markers 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

[0025] 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.

[0026] 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.

[0027] Unless otherwise specified, the hybridization techniques used in the examples are conventional hybridization techniques well known to those skilled in the art.

[0028] The rice varieties Haoxiang 24B, Wenxiang 28B, Haoxiang 24A, Wenxiang 28A, Zhuangxiang 2051A, Wenhui 184, Wenhui 206, and Wenhui 247 mentioned in the following examples are maintainer lines, sterile lines, and restorer lines bred by the Wenshan Academy of Agricultural Sciences. They are preserved in the germplasm bank of the Wenshan Academy of Agricultural Sciences and can be obtained by the public from this germplasm bank. The Yunnan local soft rice varieties Haomulü, Haomuxi, Haopi, Haomuyun, Babaogu, Haomuya, Haobayuan, Haojiehai, Haodilahao, Haoerwanghanduo, Haoanmen, Haoanong, Haogongxin, Haodadalong, Haogan, Haoha, Haoleng, Haokang, Haomuliang, and Haozhao are preserved in the germplasm bank of the Wenshan Academy of Agricultural Sciences or can be obtained from the National Germplasm Resource Bank. Yunnan's high-quality soft rice varieties, including Dianlong 201, Dianrui 408, Diantun 502, Yunhui 290, and Hongyou 5, are publicly known and widely used improved rice varieties, preserved in the germplasm bank of the Wenshan Academy of Agricultural Sciences. They can also be obtained from seed banks or breeding units. Two-line sterile lines Y58S, Long 638S, and Jing 4155S were introduced from Sichuan Longping High-Tech Seed Industry Co., Ltd.

[0029] The following examples refer to Figure 1 The breeding methods used are as described in the text.

[0030] Example 1: Screening of the soft rice donor variety Haomulü with a high-quality gray-white endosperm marker gene In this embodiment, the superior maintainer lines of fragrant soft rice with transparent endosperm, Haoxiang 24B and Wenxiang 28B, and the restorer lines Wenhui 184, Wenhui 206, and Wenhui 247, were used as male parents (as the superior rice variety II with transparent endosperm of this invention). Twenty high-quality soft rice varieties from Yunnan, including Haomulü, Haomuxi, Haopi, Haomuyun, Babaogu, Haomuya, Haobayuan, Haojiehai, Haodilahao, Haoerwanghanduo, Haoanmen, Haoanong, Haogongxin, Haodadalong, Haogan, Haoha, Haoleng, Haokang, Haomuliang, and Haozhao, were used as female parents (as the soft rice germplasm resource variety I with endosperm color specificity of this invention). Soft rice donor variety III with a high-quality grayish-white endosperm marker gene was screened. Details are as follows: In July 2004, at the Wenshan Prefecture Academy of Agricultural Sciences in Yunnan Province, 20 high-quality local soft rice varieties from Yunnan Province—including Haomulü, Haomuxi, Haopi, Haomuyun, Babaogu, Haomuya, Haobayuan, Haojiehai, Haodilahao, Haoerwanghanduo, Haoanmen, Haoanlong, Haogongxin, Haodadalong, Haogan, Haoha, Haoleng, Haokang, Haomuliang, and Haozhao—were used as female parents. The superior maintainer lines of fragrant soft rice with transparent endosperm, Haoxiang 24B and Wenxiang 28B, and the restorer lines Wenhui 184, Wenhui 206, and Wenhui 247, were used as male parents. One hundred hybrid seed combinations were obtained. In early November 2004, the hybrid seed combinations, along with their hybrid parents, were taken to the experimental base of the Wenshan Prefecture Academy of Agricultural Sciences in Sanya City, Hainan Province for planting. The experiment involved simultaneous sowing and transplanting, with each plot covering 0.01 mu (approximately 0.01 hectares). 36 clumps were transplanted per plot, with each clump containing a single plant. The F1 hybrid plants were planted adjacent to their parent plants during transplanting. In early April 2005, when the experimental materials matured, seeds from the F1 hybrids and their parent plants were harvested and taken to the rice research institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province for drying or sun-drying. When the seed moisture content dropped below 13%, the grains from each F1 hybrid and its parent plants were hulled and processed into brown rice for observation. The parent variety of the hybrid with the most segregation types of brown rice endosperm color was selected as the donor variety III for the soft rice gene with the high-quality grayish-white endosperm marker. Because the rice grains from the F1 hybrids were already F2 generation seeds, the genes controlling traits such as endosperm color had already segregated. If the endosperm color exhibited multiple types, such as transparent, semi-transparent, partially transparent, and opaque, it indicated that the control gene types for endosperm color in the parent hybrid were diverse.

[0031] The experimental results showed that the endosperm color trait of individual seeds from the F1 hybrid combinations of Haomulü / Haoxiang 24B, Haomulü / Wenxiang 28B, Haomulü / Wenhui 184, Haomulü / Wenhui 206, Haomulü / Wenhui 247, Haopi / Haoxiang 24B, Haopi / Wenxiang 28B, Haopi / Wenhui 184, Haopi / Wenhui 206, and Haopi / Wenhui 247 exhibited the greatest segregation, displaying different types such as transparent, semi-transparent, partially transparent, and opaque. The opaque portions showed varying shades of dark gray, milky white, cloudy, grayish white, silvery white, ivory, mutton fat, and jade. The experimental results proved that among this batch of Yunnan local high-quality soft rice germplasm resources I with unique endosperm color, Haomulü and Haopi contained the richest variety of genes controlling the opacity of the endosperm.

[0032] Based on routine observations, *Haomulu* has weak photosensitivity and better plant type, grain type, and resistance. Using it as a parent for hybridization, it is relatively easy to separate and select superior individual plants from the hybrid offspring. Therefore, from the brown rice of the F1 hybrid combinations such as *Haomulu* / *Haoxiang* 24B, *Haomulu* / *Wenxiang* 28B, *Haomulu* / *Wenhui* 184, *Haomulu* / *Wenhui* 206, and *Haomulu* / *Wenhui* 247 and their maternal parent *Haomulu* individual plants, after removing rice grains with transparent, semi-transparent, and partially transparent endosperm, iodine solution identification and quality physicochemical analysis tests such as amylose content, gel consistency, and gelatinization temperature were performed. Test results showed that all iodine solution reactions were blue-purple. The amylose content, gel consistency, and alkali spreading value of the opaque endosperm seeds of the F1 hybrid combinations (Haomulu / Haoxiang 24B, Haomulu / Wenxiang 28B, Haomulu / Wenhui 184, Haomulu / Wenhui 206, Haomulu / Wenhui 247, and their maternal parent, Haomulu, were within the ranges of 10.0%±0.5, 90±1.8, and 6.2±0.4, respectively. These results directly ruled out the possibility that the hybrid parents might contain glutinous rice genes, leading to hybridization and recombination resulting in opaque glutinous rice seeds (amylose content below 2%). Meanwhile, the experimental results showed that the Yunnan local high-quality soft rice variety, Haomulü, not only contains the most abundant and diverse opaque endosperm control genes, but also revealed that this opaque endosperm gene "hidden" in Haomulü may be related to, or even linked to, high-quality eating traits such as low amylose content, high gel consistency, and low gelatinization temperature, and can be stably inherited. This suggests that this heritable opaque or colored endosperm trait may be a marker trait for high-quality eating traits in rice, such as low amylose content, high gel consistency, and low gelatinization temperature. Therefore, Haomulü, with its distinctive endosperm color, was selected as the soft rice donor variety III with a high-quality grayish-white endosperm marker gene in this invention.

[0033] Example 2: Breeding of Soft Rice Line VI with High-Quality Grayish-White Endosperm Markers In this embodiment, the Yunnan local high-quality soft rice variety Haomulü, which has a unique endosperm color and was screened in Example 1, is used as the soft rice donor variety III with the high-quality gray-white endosperm marker gene in this invention. The Yunnan soft rice variety Dianrui 408, which has a good plant type, resistance to rice blast, high quality, and transparent endosperm, and carries the genetic background of Yunnan local high-quality soft rice varieties, is used as the soft rice variety IV with the genetic background of Yunnan local high-quality soft rice varieties. Soft rice single plants VI with the high-quality gray-white endosperm marker are selected and bred, specifically 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.

[0034] 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.

[0035] 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... Strict selection criteria were used to observe and eliminate four individual plants whose traits were not yet fully stable and those with weak photosensitivity. Twelve soft rice lines with grayish-white endosperm markers were successfully selected: 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. In May 2010, these twelve soft rice lines with grayish-white endosperm markers and their hybrid female parent, Haomulü, were planted adjacent to each other at the experimental base of the Wenshan Academy of Agricultural Sciences in Yunnan Province. In October 2010, when the experimental materials matured, the seeds were harvested promptly and taken to the rice research institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province for drying or sun-drying until the seed moisture content reached 13%. Then, quality physicochemical analyses were conducted, including amylose content, gel consistency, and gelatinization temperature. The results showed that the amylose content, gel consistency, and alkali spreading value of the 12 soft rice lines with grayish-white endosperm markers and their hybrid female parent, Haomulü, were all within the ranges of 10.0%±0.3, 90±1.6, and 6.1±0.3, respectively. Test results showed that 12 high-quality soft rice lines with grayish-white endosperm markers were successfully bred using grayish-white endosperm as a selection marker. Their comprehensive high-quality eating traits, such as low amylose content, high gel consistency, and low gelatinization temperature, are consistent with their hybrid maternal parent, Haomulü. This verifies that the high-quality grayish-white endosperm marker trait, which is "hidden" in the high-quality soft rice germplasm resource Haomulü in Yunnan and is related to the comprehensive excellent eating traits of rice such as low amylose content, high gel consistency, and low gelatinization temperature, can be stably inherited.

[0036] Thus far, this embodiment has selected 12 lines with high-quality gray-white endosperm phenotypic markers as the soft rice line VI with high-quality gray-white endosperm markers of the present invention.

[0037] Example 3: Breeding of Soft Rice Line VII with High-Quality Recessive Grayish-White Endosperm Markers In this embodiment, the 12 soft rice lines with high-quality grayish-white endosperm phenotypic markers selected in Example 2 are used as the soft rice single plants VI with high-quality grayish-white endosperm markers in this invention. Soft rice lines VII with high-quality recessive grayish-white endosperm markers are selected, specifically as follows: To accurately assess the yield, adaptability, disease resistance, and stability of the 12 soft rice lines with high-quality grayish-white endosperm phenotypic markers selected in Example 2, multi-location superiority identification trials were conducted in April 2011 in 11 different ecological regions, including Wenshan, Jinghong, Mengzi, Luoping, Longyang, Binchuan, and Tengchong in Yunnan Province; Guiyang in Guizhou Province; Jiangyou and Guanghan in Sichuan Province; and Nanning in Guangxi Province. Except for the pilot trial at the Wenshan Prefecture Academy of Agricultural Sciences in Yunnan Province, which used its hybrid parents as controls, all other pilot trials used its hybrid male parent, Dianrui 408, as a control. Each plot was 0.02 mu (approximately 0.02 hectares), with 360 clumps planted per plot, single-planted, without replication. At maturity, the rice varieties were evaluated 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 single-plant weight. Based on the results of the variety survey at various test sites, the yields of six lines, WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896, were all more than 5.6% higher than the control. Furthermore, these lines exhibited uniform heading, resistance to rice blast, moderate plant height, slender grains, wide adaptability, and consistent gray-white endosperm color. These findings indicate that lines WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896 are superior to the control and have good application and promotion value in production.

[0038] In November 2011, in order to accurately assess the cooking and eating qualities of six successfully bred rice lines (WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896), the six lines, along with their parent plant, Haomulü, which were being piloted at the Wenshan Prefecture Academy of Agricultural Sciences base in Yunnan Province, were selected as test samples for evaluation of cooking and eating qualities. A panel of 12 experts and managers from high-quality rice processing enterprises and agricultural technology extension units in Yunnan Province was invited to form a soft rice eating quality evaluation group. The evaluation was conducted in accordance with the national standard GB / T 15682-2008 "Sensory Evaluation Method for Cooking and Eating Quality of Rice in Grain and Oil Inspection". The results showed that the eating quality of the seven tested lines (including the control variety, Haomulü) was within the range of 96±1.2. Among them, the highest eating quality score was for line WR05-825, followed by WR05-894, and third was the hybrid maternal parent, Haomulü. The identification test results indicate that by using the high-quality grayish-white endosperm as a selection marker, these six high-yielding, highly resistant, and widely adaptable high-quality soft rice lines with the grayish-white endosperm marker were successfully bred. Their cooking quality and eating quality traits are consistent with their hybrid maternal parent, Haomulü, further confirming that the cooking quality and eating quality traits of soft rice with the high-quality grayish-white endosperm marker can also be stably inherited.

[0039] Meanwhile, to accurately identify the genetic relationship between the high-quality grayish-white endosperm marker trait and the high-quality transparent endosperm trait, in April 2012, at the experimental base of the Wenshan Academy of Agricultural Sciences in Sanya City, Hainan Province, six lines (WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896) were selected as soft rice lines with the high-quality grayish-white endosperm marker. Six superior Yunnan soft rice varieties (Dianlong 201, Wenhui 206, Wenhui 247, Hongyou 5, Yunhui 290, and Diantun 502) were selected as high-quality transparent endosperm soft rice varieties. Pairwise reciprocal hybridization was conducted to obtain 72 hybrid seed combinations. These were then dried or sun-dried in the storage room of the Rice Research Institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province. When the seed moisture content dropped to 13%, the endosperm color of the hybrid seeds was observed after shelling. By May 2012, 72 F1 plants of hybrid combinations were planted at the experimental base of the Wenshan Academy of Agricultural Sciences in Yunnan Province. Each plot contained 36 transplanted clumps, with a single plant per clump, and no replicates. In October 2012, when the experimental materials matured, five true hybrid plants were selected from the middle row and taken to the rice research institute of the Wenshan Academy of Agricultural Sciences in Yunnan Province for drying or sun-drying until the seed moisture content dropped to 13%. The segregation of endosperm color traits in the seeds of each F1 hybrid combination was then observed, and the result was calculated using a chi-square test. 2The fitness test inferred the genetic pattern of segregation of the gray-white endosperm phenotypic markers.

[0040] The experimental results showed that, regardless of whether it was a reciprocal cross or a cross, the endosperm of the contemporary seeds of the hybrid combinations of high-quality transparent endosperm soft rice varieties and high-quality gray-white endosperm soft rice lines were all transparent. The segregation ratio of the high-quality transparent endosperm trait to the high-quality gray-white endosperm soft rice marker trait was 3:1, indicating that the high-quality transparent endosperm soft rice trait is dominant over the high-quality gray-white endosperm soft rice trait, and the high-quality gray-white endosperm soft rice marker trait is controlled by one pair of recessive genes.

[0041] Thus, in this embodiment, six strains, namely WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896, were selected as the soft rice strain VII of this invention with a high-quality recessive gray-white endosperm marker.

[0042] Example 4: Breeding of Wenbaihui 825, a soft-grained rice variety with a high-quality recessive gray-white endosperm marker. This embodiment uses six lines selected in Example 3—WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, and WR05-896—as the soft rice line VII with a high-quality recessive gray-white endosperm marker, and selects and breeds a soft rice variety IX with a high-quality recessive gray-white endosperm marker. Details are as follows: In April 2013, six successfully bred soft rice lines VII with high-quality gray-white endosperm markers were used as test varieties, and Hongyou 6, the then-control variety for conventional high-quality rice in Yunnan Province, was used as the control variety for variety comparison and demonstration. The variety comparison trial was conducted at the Wenshan Prefecture Academy of Agricultural Sciences experimental base in Yunnan Province, with each plot covering 0.02 mu (approximately 0.02 hectares), planting 400 clumps per plot, single-plant planting, and three replicates. When the test materials matured in September 2013, a variety evaluation survey was conducted 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 three lines, WR05-825, WR05-896, and WR05-882, were more than 5.2% higher than the control variety, Hongyou 6. The highest yield was achieved by line WR05-825, reaching 588.6 kg / mu, a 6.1% increase over Hongyou 6, and it exhibited the best yield characteristics and strongest resistance. High-yield cultivation demonstration sites were set up in ecological rice-growing areas at different altitudes in Wenshan, Malipo, and Funing in Wenshan Prefecture, and Mangshi in Dehong Prefecture. Hongyou 6, a fragrant soft rice variety widely promoted in Yunnan Province at the time, was used as the control. Each line was planted at 0.8 mu per demonstration site, managed according to the local rice planting and management standards. The results showed that only two lines, WR05-825 and WR05-882, achieved an average yield more than 5.0% higher than the control across all demonstration sites, with yields of 576.4 kg / mu and 558.2 kg / mu, respectively. The WR05-825 strain had the highest yield, the strongest resistance to rice blast, and good color change after maturity at all demonstration sites.

[0043] To accurately assess the combining ability of the successfully bred soft rice line VII, which possesses a high-quality recessive gray-white endosperm marker, in April 2013, at the Wenshan Academy of Agricultural Sciences experimental base in Sanya City, Hainan Province, the following hybridization was conducted: using the three-line male-sterile soft rice lines with good transparency (Haoxiang 24A, Wenxiang 28A, Zhuangxiang 2051A) and the two-line male-sterile lines (Y58S, Long 638S, Jing 4155S) as female parents, and using six soft rice lines with a high-quality recessive gray-white endosperm marker (WR05-825, WR05-868, WR05-882, WR05-890, WR05-894, WR05-896) selected in Example 3 as male parents, pairwise hybridization was performed, yielding 36 test-matching combinations. In May 2013, these 36 test-matching combination seeds were planted at the Wenshan Academy of Agricultural Sciences experimental base in Yunnan Province. Each experimental plot covered 0.02 mu (approximately 0.02 hectares), with 360 clumps planted per plot. Single-plant planting was implemented without replication. The control variety, II You 838, from the then-current regional trials of high-quality hybrid rice in Yunnan Province, was used as the control. In September 2013, when the experimental combinations 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 weight.

[0044] The test results showed that eight tested combinations, including Wenxiang 28A / WR05-825, Jing 4155S / WR05-825, Haoxiang 24A / WR05-825, Zhuangxiang 2051A / WR05-825, Y58S / WR05-825, Wenxiang 28A / WR05-882, Haoxiang 24A / WR05-896, and Jing 4155S / WR05-896, had higher yields than the control. Among them, Wenxiang 28A / WR05-825 and Jing 4155S / WR05-825 increased yields by more than 5.0% compared with the control, with yields of 636.7 kg / mu and 614.2 kg / mu, respectively. The experiment showed that the WR05-825 strain not only has the strongest combining ability, but also, when used as the male parent and tested with multiple sterile lines, the yield of most of the test combinations was higher than that of the control, demonstrating strong combining ability and good application prospects as a restorer line.

[0045] Because the WR05-825 variety exhibits superior quality, highest yield, strongest combining ability, strong resistance, and high productivity in both taste and cooking quality assessments, as well as in comparative and crossbreeding combination advantage assessments, especially given its bright and uniform gray-white endosperm and single genetic trait, WR05-825 is named Wenbaihui 825, which is the soft rice variety IX of this invention with a high-quality recessive gray-white endosperm marker.

[0046] In summary, the breeding method of this invention has the following advantages: 1. The seed selection of this invention is not limited by the hybridization segregation generation, and has high precision and strong targeting, which reduces the cost of high-quality rice breeding and shortens the breeding cycle; Specifically, the inventors successfully discovered a gene with a high-quality recessive gray-white endosperm soft rice marker trait from high-quality local soft rice germplasm resources in Yunnan. This gene was introduced into superior varieties through hybridization and other methods. Through allelic recombination of this marker trait, a homozygous high-quality recessive gray-white endosperm soft rice marker trait genotype was formed. Therefore, seeds with the high-quality recessive gray-white endosperm soft rice marker (F2 generation seeds) were selected from the F1 generation of hybrid segregated individuals for further generation. All hybrid offspring were single grains and single plants with the high-quality recessive gray-white endosperm soft rice marker. Using the high-quality recessive gray-white endosperm soft rice phenotypic trait as the selection marker for further generation, the influence of the hybrid segregation generation can be eliminated. From the F1 generation onwards, seeds can be dehydrated and dried, and observed after hulling to directly select single grains and single plants with the high-quality recessive gray-white endosperm soft rice marker trait, until a high-quality, high-yield, and multi-resistant rice variety is successfully bred. Meanwhile, this method uses only the high-quality recessive gray-white endosperm soft rice phenotypic trait as a marker. From F1 to each segregating generation of the selected variety, no special instrumental equipment is required for detection. This allows for precise selection of high-quality single grains and plants. Essentially, from F1 onwards, by using the high-quality recessive gray-white endosperm soft rice phenotypic trait as a selection marker, the selected single grains and plants are all of excellent quality soft rice. Therefore, compared with currently reported methods for high-quality rice breeding, especially soft rice breeding, this method is not only accurate, fast, simple, and practical, but also highly targeted. It allows 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.

[0047] 2. This invention is the first to systematically discover high-quality soft rice germplasm resources with high-quality recessive gray-white endosperm markers from high-quality soft rice germplasm resources in Yunnan, which enriches the types of high-quality rice and meets the market's diversified demand for high-quality rice. Specifically, addressing the challenges of high-quality rice breeding, particularly high-quality soft rice, and the changing market demand for high-quality rice with transparent endosperm, the inventors pioneered the idea of ​​discovering new high-quality soft rice varieties with high-quality colored endosperm soft rice markers from Yunnan's local high-quality soft rice germplasm resources. They led a research project on breeding high-quality soft rice with high-quality colored endosperm soft rice markers, and after nearly twenty years of effort, successfully discovered high-quality soft rice gene resources with high-quality recessive gray-white endosperm markers from Yunnan's local high-quality soft rice germplasm resources. Using these as gene donor varieties, and through hybridization and the use of high-quality recessive gray-white endosperm soft rice as selection markers, they successfully bred the high-quality soft rice variety Wenbaihui 825, filling the gap in my country's indica rice varieties that lacked new high-quality soft rice varieties with high-quality recessive gray-white endosperm markers. The promotion and development of these high-quality soft rice varieties with high-quality gray-white endosperm markers will play a significant role in meeting the increasing and diversified demands for high-quality rice as people's living standards improve.

[0048] 3. This invention utilizes high-quality local soft rice germplasm resources in Yunnan to successfully discover high-quality soft rice gene resources with high-quality recessive gray-white endosperm markers. The discovery and utilization of materials are extensive, the discovery method is simple, and the cost is low. Specifically, the inventors proposed and implemented a research project on breeding high-quality soft rice with a high-quality recessive colored endosperm marker. A new germplasm resource with a high-quality recessive gray-white endosperm marker was successfully discovered from Yunnan local soft rice germplasm resources and used as a donor for the high-quality soft rice marker gene. This gene was introduced into improved rice varieties, and using the high-quality recessive colored endosperm as a selection marker, a new high-quality soft rice variety, Wenbaihui 825, with a high-quality recessive gray-white endosperm marker was quickly and easily bred. This lays a solid foundation of germplasm genetic materials for high-quality rice endosperm marker breeding in my country and further enriches the types of colored endosperm markers and high-quality soft rice varieties in my country. Therefore, compared with currently reported international methods for high-quality rice breeding, especially soft rice breeding, this method utilizes Yunnan local high-quality soft rice germplasm resources to discover the high-quality recessive colored endosperm soft rice marker gene, with a wide range of material sources, a simple and practical discovery method, and lower costs.

[0049] 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.

[0050] 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 soft-grained rice with a latent grayish-white endosperm marker, characterized in that, Includes the following steps: 1) A soft rice germplasm resource variety I with a specific endosperm color was crossed with a rice variety II with transparent endosperm. After screening, a soft rice donor variety III with a gray-white endosperm marker gene was obtained. 2) The soft rice donor variety III and the soft rice improved variety IV were crossed to obtain hybrid seed V. The gray-white endosperm was used as a breeding marker. After multiple self-pollination generations of the F1 generation of the hybrid seed V, b soft rice lines VI with gray-white endosperm marker were obtained. 3) The characteristics of the soft rice line VI and the genetic traits of the gray-white endosperm marker were tested to obtain c soft rice lines VII with recessive gray-white endosperm markers controlled by a recessive single gene; 4) The yield of the soft rice line VII was tested, and the combining ability was identified after hybridization with the male-sterile line VIII to obtain soft rice IX with a recessive gray-white endosperm marker; Where a, b, and c are positive integers; In step 1), the soft rice germplasm resource variety I is from Yunnan local soft rice germplasm resources and its derivative varieties; Yunnan local soft rice germplasm resources and its derivative varieties include Haomulü; the screening includes selecting the maternal variety of the hybrid combination with the most segregation types of brown rice endosperm color traits as the soft rice gene donor variety III with high-quality gray-white endosperm marker; In step 1), the soft rice donor variety III with the gray-white endosperm marker gene is Haomulü, and the preservation number of Haomulü is CCTCC NO: P202407.

2. The method for breeding soft rice with a latent gray-white endosperm marker according to claim 1, characterized in that, The soft rice with the recessive gray-white endosperm marker refers to soft rice whose brown rice endosperm is gray-white after the rice grains have matured and dried, with an amylose content of 9.5-10.5%, a gel consistency ≥90mm, and an alkali spreading value ≥6.0; and the recessive gray-white endosperm marker is controlled by a single recessive gene and can be stably inherited.

3. The method for breeding soft rice with a latent gray-white endosperm marker according to claim 1, characterized in that, In step 1), the endosperm color specificity refers to the different endosperm colors among different grains in the same ear and among different individual plants; the endosperm color specifically includes: transparent and / or opaque; the opaque color includes one or more of dark gray, milky white, cloudy, grayish white, silver white, ivory, mutton fat and jade, and the endosperm color specificity varies greatly among different soft rice germplasm resource varieties I.

4. The method for breeding soft rice with a latent gray-white endosperm marker according to claim 1, characterized in that, In step 1), the rice variety II with transparent endosperm is a rice variety with endosperm transparency reaching level 3 or above of the national standard, and which does not contain photosensitive genes, has a short growth period, good high-yield traits, and strong resistance.

5. The method for breeding soft rice with a latent gray-white endosperm marker according to claim 1, characterized in that, In step 2), the soft rice superior variety IV is obtained by hybridization and selection using the soft rice germplasm resource variety I with the endosperm color as the parent; the number of generations of self-pollination is 6-8.

6. The method for breeding soft rice with a latent gray-white endosperm marker according to claim 1, characterized in that, In step 3), the characteristics of the soft rice strain VI include yield, resistance, adaptability and stability.

7. The application of the soft rice breeding method with a latent gray-white endosperm marker as described in any one of claims 1-6 in rice breeding.

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