Establishment method of rice nuclear three-line breeding system and application of rice nuclear three-line breeding system in breeding

Abstract

The invention relates to the field of rice breeding, and provides an establishment method of a rice nuclear three-line breeding system and a corresponding breeding method. The inventor of the application finds that a new material F1312 has a very unique fertility characteristic in the test cross process of the restorer line, that is, the material is fertile under high and low temperature conditions, the fertility does not change along with environmental conditions, but the fertility can generate specific change through hybridization with the other two types of materials. Genetic localization finds that the character is controlled by a single gene, and the character is named as HS10. On the basis, the invention proposes to establish a nuclear three-line system, that is, near-isogenic line materials alpha line and beta line which are consistent in genetic background, have no obvious difference in agronomic traits and respectively carry alpha and beta alleles at the HS10 site are bred, hybridization of the alpha line and the beta line is realized by combining with other sterile systems, and hybrid male sterile alpha and beta lines are produced on a large scale. And matching and hybrid rice seed production are carried out by taking a material carrying R alleles as a restorer line (R line), so that the purpose of utilizing heterosis is achieved.

Description

Technical Field

[0001] The present invention relates to the field of crop breeding, and specifically to a method for establishing a "nuclear three-line" breeding system for rice and its application in breeding. The method of the present invention can utilize heterosis in breeding. Background Art

[0002] Heterosis refers to the phenomenon that the phenotypic values of biomass, growth rate, and yield of the hybrid offspring of different varieties or species of a species are superior to those of the two parents. Rice is one of the most important food crops in China, and more than 60% of the population in China mainly eats rice. Rice is a natural self-pollinated crop. In order to utilize heterosis, it is necessary to create sterile lines for large-scale hybrid seed production. The invention and application of hybrid rice have made great contributions to food security in China and even the world.

[0003] In 1966, Shinjo Chohyuku crossed Tsai-chung 65 with Chinsurah Boro II to develop the Tsai-chung 65 sterile line, achieving the three-line matching of japonica rice (referred to as cytoplasmic male sterility of the Boro type, CMS-BT), but the heterosis was not obvious. In 1970, Mr. Yuan Longping and his assistant Li Bihu et al. discovered pollen-aborted wild rice and sterile cytoplasm in Sanya, Hainan (referred to as cytoplasmic male sterility of the wild abortive type, CMS-WA), opening a breakthrough for the breeding of indica hybrid rice male sterile lines. In addition to CMS-WA, Chinese rice breeders have also developed the Gang type, D type, K type, dwarf abortive type, and Indonesian paddy type. The sterility characteristics and restorer and maintainer relationships of these sterile lines are similar to those of the wild abortive type and all belong to sporophyte sterility. In 1972, Mr. Zhu Yingguo of Wuhan University et al. crossed red-mangrove wild rice with Liantangzao to select the Honglian type cytoplasmic male sterile line (CMS-HL).

[0004] The pollen abortion of the BT type and HL type sterile lines belongs to gametophyte sterility, and the sterility is not as stable as that of CMS-WA. It is easy to self-pollinate and set seeds at high temperatures. Moreover, among the existing indica rice resources, only 0.1% can be transferred into three-line sterile lines or maintainer lines, and only 5% can be used as three-line restorer lines. Deficiencies such as unfree combination and low resource utilization rate limit the exertion of heterosis in three-line hybrid rice.

[0005] In 1973, Mr. Shi Mingsong discovered the photoperiod-sensitive genic male-sterile (PGMS) mutant Nongken 58S in Nongken 58, and proposed the idea of using male sterility under long-day and high-temperature conditions for seed production and fertility under short-day and low-temperature conditions for reproduction, with one line having two uses. Subsequently, a number of thermo-sensitive genic male-sterile lines with male sterility under high temperature and fertility under low temperature (such as Annong S-1, Hengnong S-1, and 5460S, etc.) and reverse thermo-sensitive male-sterile lines with male fertility under high temperature and male sterility under low temperature (such as Yannong S) were discovered and applied. In theory, 95% of the existing rice resources can be transformed into two-line male-sterile lines or restorer lines, which breaks through the restriction of the restorer-maintainer relationship of cytoplasmic-nuclear interaction three-line on the utilization of germplasm resources, greatly increasing the probability of breeding excellent combinations.

[0006] However, the sterility of two-line male-sterile lines is easily affected by light and temperature conditions, resulting in great risks in the seed production of two-line hybrid rice, and frequent failures in the seed production of two-line hybrid rice. Since the sterility starting temperature is controlled by minor polygenes, the genetic complexity determines that it is not easy to breed two-line male-sterile lines with a low sterility starting temperature.

[0007] Therefore, cultivating new male-sterile lines with safe fertility, free combination ability, and high resource utilization rate is of great significance for promoting the development of hybrid rice.

[0008] Liang Manzhong et al. (2006) crossed the recessive genic male-sterile rice go543S (Yannong S) with low-temperature-induced pollen sterility with the photoperiod-thermo-sensitive genic male-sterile rice Nongken 58S and the photoperiod-thermo-sensitive genic male-sterile lines 7001S, Peiai 64S, and Changxuan 3S derived from Nongken 58S, and found that the pollen of the hybrid F1 was sterile under natural long-day and high-temperature, short-day and low-temperature, and different artificial light and temperature treatment conditions; in the backcross combinations, the proportion of sterile plants with a fertility conversion period like the male parent was about 50%, and the proportion of permanently sterile plants like the hybrid F1 was about 50%, conforming to the 1:1 segregation ratio of one pair of recessive genes in backcross.

[0009] Ni Jinlong et al. (2019, 2022, and 2023) further studied this trait of Yannong S, mapped the gene to chromosome 10, and proposed to use three allelic types rtms10 L422 、rtms10 YNS and rtms10 1892F at this locus to design a reproducible heterozygous male-sterile line. In this system, when the allelic type rtms10 YNS is crossed with rtms10 1892F , F 1 shows heterozygous sterility, and rtms10 L422 can restore the fertility of the heterozygous male-sterile line. However, this system is restricted by the reverse thermo-sensitive male-sterility gene rtms10 YNS , and contains rtms10 YNSThe materials of the allele type exhibit the phenotype of low-temperature-induced pollen sterility similar to Yanong S. Using the rtms10 YNS type material as the female parent to reproduce the heterozygous sterile line is only limited to the areas with lower temperatures, and the reproduction space is restricted. If using the rtms10 YNS type material as the male parent and rtms10 1892F as the female parent, it can be reproduced in high-temperature areas. However, due to the sterile characteristic of rtms10 YNS itself, even under the condition of becoming fertile at high temperatures, the pollen amount is not as sufficient as that of the conventional fertile materials, which easily leads to a low reproduction yield of the sterile line. SUMMARY OF THE INVENTION

[0010] Therefore, in the existing breeding systems, there are certain problems respectively. In view of the above problems, the present invention provides a new "nuclear three-line" breeding system and the corresponding breeding method.

[0011] PRINCIPLE OF THE INVENTION

[0012] The inventor of the present application found a new material F1312 during the test cross of the restorer line. The inventor found that this material has very unique fertility characteristics. It is a fertile material under both high and low temperature conditions, and its fertility does not change with the environmental conditions. However, its fertility can undergo specific changes through hybridization with two other types of materials. Therefore, the inventor conducted a large number of breeding experiments on this material and deposited it in the China General Microbiological Culture Collection Center. Rice Oryzasativa, deposit date: 2023-11-23, deposit number CGMCC NO.: 45697.

[0013] Specifically, the inventor found that when F1312 was hybridized with 1892S (a thermosensitive genic male sterile line) and 1892F (fertile under both high and low temperatures), the seed setting rate of the F1 generation was very low. Using 1892F as the recurrent parent, the sterile plants and fertile plants in the backcross progeny showed a segregation ratio of 1:1. The BSA method located this locus on chromosome 10 and named it HS10, as shown in Figure 1 .

[0014] The invention divides HS10 into three allele types, namely α (such as 1892F, etc.), β (such as F1312, etc.) and R type (such as Guangzhan 63S, etc.). The rice materials containing α, β and R allele types are respectively named α line (genotype: αα), β line (genotype: ββ) and R line (genotype: RR). Among them, when the α line and the β line are hybridized, the F1 generation (genotype: αβ) shows a male sterile phenotype (pollen is not stained by iodine-potassium iodide solution under low temperature conditions, and some pollen can be stained by iodine-potassium iodide solution under high temperature conditions, but it cannot self-pollinate and set seeds or the seed setting rate is lower than 0.5%). When the F1 generation is hybridized with the R line, the offspring all recover fertility and set seeds normally.

[0015] On this basis, the present invention proposes to establish a "nuclear three-line" system for hybrid rice, that is, to breed near-isogenic line materials with the same genetic background, no obvious difference in agronomic traits, and carrying α-type and β-type alleles at the HS10 locus respectively. The α-line and the β-line are hybridized to produce a heterozygous male sterile line αβ-line. Using the material carrying the R allele as the restorer line (R-line), and the αβ-line as the sterile line, to carry out mating and hybrid rice seed production, so as to achieve the purpose of utilizing the heterosis of rice (such as Figure 2 ). It should be noted that the above HS10 locus refers to the interval of 13204998-13505990 on chromosome 10 of rice with 93-11 as the reference genome (http: / / www.elabcaas.cn / rice / index.html).

[0016] Furthermore, a specific breeding method for the α-line and the β-line that are near-isogenic lines to each other is proposed. If the target rice is of the R type, using the R-type target rice as the recurrent parent, and respectively backcrossing and self-crossing with the α-type and β-type rice, and through molecular marker selection, finally breeding the near-isogenic line α-line and β-line with the target rice as the genetic background; if the target rice is of the α type, using the α-type target rice as the recurrent parent, hybridizing with the β-type and R-type rice respectively and then continuously backcrossing until the agronomic traits are stable, then using the sterile αβ single plant as the female parent and the fertile αR single plant as the male parent to hybridize, screening the single plant with the genotype βR by molecular markers, harvesting the seeds for planting, and then screening the single plant with the genotype ββ by molecular markers, finally breeding the β-line with the target rice as the background; similarly, if the target rice is of the β type, the α-line can be bred.

[0017] The α-line and β-line of the present invention can be combined with other sterile systems (such as the two-line positive photoperiod-temperature sensitive or reverse photoperiod-temperature sensitive system) for large-scale production of the heterozygous male sterile αβ-line. For example, let the α-line carry the two-line positive thermosensitive sterile gene tms5, etc., to breed the αS-line, which is male sterile under high temperature conditions (such as in summer in the middle and lower reaches of the Yangtze River and the South China rice regions), and can be hybridized with the β-line (fertile at high and low temperatures) to produce the αβ-line (male sterile at high and low temperatures); under low temperature conditions (such as in winter in Hainan), the αS-line turns into male fertile and can be normally propagated. On the contrary, breeding the βS-line and mating with the α-line (fertile at high and low temperatures) can also realize the matching of the "nuclear three-line".

[0018] At the same time, the present invention also proposes a breeding method for the restorer line in the "nuclear three-line" system, that is, using molecular marker selection to select the rice with the R type at the HS10 locus as the restorer line; or selecting the rice with a seed setting rate of more than 70% in the offspring of hybridization with the αβ-line as the restorer line.

[0019] Specifically, the present invention provides a method for establishing a "nuclear three-line" breeding system for rice, and near-isogenic line materials α-line and β-line carrying α allele type and β allele type at the HS10 locus are developed. Among them, the β-line is a rice material that is fertile at both high and low temperatures, and the α-line is a rice material that is sterile at both high and low temperatures after crossing with the β-line. Under the sterile condition of the sterile gene, the α-line is crossed with the β-line to produce αβ-type heterozygous sterile line seeds αβ, and then the heterozygous sterile line αβ is crossed with the R-type restorer line to produce hybrid rice seeds. The R-type restorer line is a rice material that restores fertility after crossing with the heterozygous sterile line αβ.

[0020] In a preferred implementation manner, the HS10 locus refers to the interval of 13204998-13505990 on chromosome 10 of rice with 93-11 as the reference genome.

[0021] In another preferred implementation manner, the β-line is F1312 and its derivatives. The α-line carrying the α allele type includes rice varieties 1892S, 1892F and their derivatives. The β-line carrying the β allele type includes rice variety F1312 and its derivatives. The R-line carrying the R allele type includes Guangzhan 63S, Huazhan, R0822, HK57 and their derivatives.

[0022] In another preferred implementation manner, for the Indel-type molecular marker for detecting α, β and R allele types, the primers are the sequences shown in SEQ ID NO.1-SEQ ID NO.10.

[0023] In another preferred implementation manner, the sterile gene includes the sterile gene carried in the two-line positive photo-thermo-sensitive system or the reverse photo-thermo-sensitive system.

[0024] In another preferred implementation manner, the sterile gene of the two-line positive photo-thermo-sensitive system includes tms5. The method includes introducing the tms5 gene into the α-line rice plants to develop the αS-line, which is male sterile under high temperature conditions and turns male fertile under low temperature conditions. The αS-line is crossed with the β-line to produce the αβ-line, which is male sterile at both high and low temperatures; or introducing tms5 into the β-line plants to develop the βS-line, and crossing the βS-line with the α-line plants that are fertile at both high and low temperatures to produce the αβ-line.

[0025] In another preferred implementation manner, the allele types carried by the α-line and the β-line can be interchanged with each other.

[0026] On the other hand, the present invention provides a rice breeding method, which judges the type of the target rice variety, determines the type of the allele at the HS10 locus of the target rice, and classifies the target rice variety into α-type, β-type or R-type; respectively selects the α-line and the β-line according to different alleles of the target rice at the HS10 locus:

[0027] If the target rice is of the R type, using the R-type target rice as the recurrent parent, backcross it with α-type and β-type rice respectively. Through molecular marker selection, select individual plants with genotypes αR and βR respectively for continuous backcrossing until the agronomic traits of the high-generation are stable, and then self-cross. Use molecular marker selection to obtain near-isogenic line materials with genotypes αα and ββ, and finally cultivate the α line and β line with the target rice as the genetic background. If the target rice is of the α type, using the α-type target rice as the recurrent parent, hybridize it with β-type and R-type rice respectively and then conduct continuous backcrossing. During the hybridization and backcrossing with the β line, the individual plants with the genotype αβ are male sterile. Select the male sterile individual plants to continue backcrossing with the target material. At the same time, cross the target material with the R line, and use molecular marker-assisted selection to select individual plants with the genotype αR for continuous backcrossing until the agronomic traits of the backcross offspring are stable. Subsequently, use the sterile αβ individual plants as the female parent and the fertile αR individual plants as the male parent for hybridization, screen the individual plants with the genotype βR by molecular markers, harvest the seeds, plant F2, and then screen out the individual plants with the genotype ββ by molecular markers to finally cultivate the β line with the target rice as the background.

[0028] Similarly, if the target rice is of the β type, hybridize the β-type target rice with the α line and the R line respectively and then conduct continuous backcrossing. Use molecular marker-assisted selection, and then through the hybridization and self-crossing of the sterile αβ line and the fertile βR line, cultivate the α line with the target rice as the background.

[0029] On the other hand, the present invention provides a breeding method for the restorer line in a "nuclear three-line" breeding system, which is characterized in that using molecular marker selection, selecting rice with the HS10 locus being of the R type as the restorer line; or selecting rice with a seed setting rate of more than 70% in the offspring of hybridization with the αβ line as the restorer line.

[0030] Those skilled in the art should understand that although the description in the embodiments of the present invention is based on F1312, other rice materials that are fertile at high and low temperatures are equally applicable according to the method of the present invention.

[0031] Beneficial effects

[0032] The present invention provides a "nuclear three-line" breeding system and corresponding breeding methods with a simple genetic model and not restricted by the starting temperature of the two-line system and the thermosensitive gene. The breeding system of the present invention has a high resource utilization rate and can make more full use of heterosis. Description of the drawings

[0033] Figure 1 : HS10 chromosome localization map

[0034] Figure 2 : "Nuclear three-line" pattern diagram

[0035] Figure 3 : Process of breeding the α line and β line from the R-type material

[0036] Note: If the target material is a two-line sterile line, then α-type and β-type rice can be selected as fertile materials; if the target material is a conventional fertile material, then one of the α-type and β-type can be a two-line sterile line. The genotypes are shown in parentheses. MAS is marker-assisted selection. While selecting the α, β, and R allelic types, the two-line sterile gene can also be selected.

[0037] Figure 4 : Process of breeding β line from α-type material

[0038] Note: If the target material is a two-line sterile line, then β-type and R-type rice can be selected as fertile materials; for the cross between an αβ single plant and the α line, it needs to be carried out when the α line is in the fertile state. If the α line is a conventional fertile material, then one of the β-type and R-type materials can be a two-line sterile line. The genotypes are shown in parentheses. MAS is marker-assisted selection. While selecting the α, β, and R allelic types, the two-line sterile gene can also be selected.

[0039] Figure 5 : Process of breeding α line from β-type material

[0040] Note: If the target material is a two-line sterile line, then α-type and R-type rice can be selected as fertile materials; for the cross between an αβ single plant and the β line, it needs to be carried out when the β line is in the fertile state. If the β line is a conventional fertile material, then one of the R-type and α-type rice can be a two-line sterile line. The genotypes are shown in parentheses. MAS is marker-assisted selection. While selecting the α, β, and R allelic types, the two-line sterile gene can also be selected. Specific implementation manners

[0041] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention belongs to the scope of the present invention. If not specifically specified, the technical means used in the examples are conventional means well-known to those skilled in the art.

[0042] I. Discovery of heterozygous male sterility

[0043] In 2014, during the test cross process, the inventor discovered a resource F1312 (source: RH003 / / / / Wuhu 26 / / / RH003 / / Guangzhan 63S / Xa23, Pi9 and BT pyramiding line, self-crossed for the 6th generation, and has been deposited in the China General Microbiological Culture Collection Center, deposit number CGMCC NO.: 45697). The pollen of this resource is normally fertile at high and low temperatures, and the seed setting is normal. However, in the F1 generation of the test cross with 1892S (under natural conditions), the anthers are small and the seed setting is very poor. To exclude the possible influence of the two-line sterility gene in the 1892S genome on seed setting, using 1892F (fertile near-isogenic line of 1892S, which is a stable material that is fertile at high and low temperatures and was developed by backcrossing 6 generations and self-crossing 4 generations with 1892S as the recurrent parent and Guanghui 102 as the fertile gene donor) as the female parent and F1312 as the male parent, the F1 generation of the cross still showed the phenotype of small anthers and very poor seed setting; to exclude the influence of cytoplasm, using F1312 as the female parent and 1892F as the male parent, the F1 of the cross still showed the male sterile phenotype. At the same time, using the F1 of the cross between 1892F and F1312 as the female parent and 1892F or F1312 as the male parent for backcrossing, the female parent can set seeds normally; but using 1892F or F1312 as the female parent and the F1 of the cross between 1892F and F1312 as the male parent for backcrossing, no hybrid seeds can be obtained. This indicates that the poor seed setting of the F1 of the cross between 1892F and F1312 is caused by male sterility.

[0044] II. Genetic analysis and gene mapping of heterozygous sterility

[0045] To study the genetic law of heterozygous sterility caused by F1312, using 1892F as the female parent and F1312 as the male parent for crossing, and then backcrossing the F1 generation with 1892F to construct BC 1 F 1 、BC 2 F 1 、BC 3 F 1 and BC 4 F 1 generation segregation populations, counting the number of sterile and fertile plants in the populations, and performing chi-square tests. It was found that the segregation ratio of male sterile and fertile plants in the 4 backcross generations conformed to 1:1 (Table 1), indicating that this heterozygous sterility inheritance is controlled by a single locus and the segregation ratio is not affected by environmental conditions.

[0046] Respectively take the leaves of fertile and sterile single plants in the BC 2 F 1 generation, extract DNA, construct a fertile pool and a sterile pool, perform genome resequencing, and map the locus of the heterozygous sterility gene by the BSA method. The results are as Figure 1 . As Figure 1 known, the heterozygous sterility gene is located on chromosome 10, and this locus is named HS10( Hheterozygous S sterile gene on chromosome 10 )。Further, BC 2 F 1 The populations were pollinated in a mixed manner, and the seeds set on the sterile plants were harvested. The segregating populations were then planted, and the phenotypes of each plant were identified through phenotypic identification. HS10 was mapped to the interval chr10:13204998-13505990 (93-11 reference genome, http: / / www.elabcaas.cn / rice / index.html) by positional cloning method.

[0047] Table 1: Statistics and chi-square test of sterile and fertile plants in each backcross population

[0048] Population Observation location Number of sterile plants Number of fertile plants Theoretical segregation ratio P-value <![CDATA[BC 1 F 1 > Lingshui 11 9 1:1 0.655 <![CDATA[BC 2 F 1 > Hefei 22 19 1:1 0.639 <![CDATA[BC 3 F 1 > Lingshui 11 11 1:1 1.000 <![CDATA[BC 4 F 1 > Hefei 198 221 1:1 0.261

[0049] Note: The P-value of the 4 populations is greater than 0.05, indicating that the chi-square test conforms to the segregation ratio of 1:1.

[0050] III. Determination of three allelic types

[0051] The allelic type of 1892S or 1892F at the HS10 locus was named α type, and the material with the genotype αα was named α line; the allelic type of F1312 at this locus was named β type, and the material with the genotype ββ was named β line; when the α line and the β line were crossed, the F1 generation (genotype αβ, also known as the αβ line) showed a male sterile phenotype. It should be noted that although in this example, the allelic type of 1892S or 1892F at the HS10 locus was named α type, those skilled in the art should understand that any rice material that is sterile at high and low temperatures after crossing with the β line can be used as the α type. To determine the α line, those with a homologous sequence with 1892S or 1892F at the HS10 locus can be defined as the α line by gene sequencing, or the fertility can be judged after crossing with the β line, and any variety that is sterile at high and low temperatures can be used as the α line.

[0052] Using Guangzhan 63S as the female parent to test-cross F1312, F 1 The seed setting rate was normal under both high temperature (normal season in Hefei) and low temperature (winter in Hainan) conditions, reaching over 85%. Using Guangzhan 63S as the female parent and 1892F as the male parent, the F 1 generation could set seeds normally under both high temperature (normal season in Hefei) and low temperature (winter in Hainan) conditions. In Hainan, using the F 1 (male sterile) of 1892F / F1312 as the female parent and Guangzhan 63S as the male parent, all the offspring plants were fertile under both high temperature and low temperature conditions.

[0053] Furthermore, among the introgression line materials with Guangzhan 63S as the donor and 1892S as the recurrent parent after three generations of backcrossing, when detected with molecular markers at the HS10 locus, three of the lines contained the Guangzhan 63S allele type. Then, three α-type (1892S-type) plant lines were randomly selected, and a total of six plant lines were crossed with F1312. As a result, when the three plant lines containing the Guangzhan 63S allele type were crossed with F1312, the seed setting rate returned to normal (above 85%), while the anthers of the hybrid F1 generation of the α-type plant lines were small and showed male sterility, and the seed setting rate was all below 25% (in the natural state).

[0054] This indicates that the Guangzhan 63S allele type at the HS10 locus can restore fertility when crossed with α-type, β-type, and αβ-type respectively, enabling normal seed setting in the hybrid offspring. The allele type that can restore the fertility of the male-sterile αβ line to normal is named the R type ( R estore), and the material with the genotype RR is called the R line.

[0055] IV. Development of Polymorphic Markers at the HS10 Locus

[0056] The second-generation genomic resequencing was performed on 1892S, F1312, and Guangzhan 63S, and polymorphic primers were designed based on the sequence differences in the vicinity of the HS10 locus in the genome. The marker information is shown in Table 2.

[0057] Table 2 Polymorphic Marker Information

[0058]

[0059]

[0060] The above primers were used to perform PCR amplification on the materials, with 1892S, F1312, and Guangzhan 63S or materials of the same type as the control. The PCR reaction system was as follows: 2 μl of genomic DNA at 20 ng / μl; 2 μl of 5×G0Taq Flexi buffer; 0.5 μl each of the upstream and downstream primers at a concentration of 10 mM; 1 μl of dNTPs at 2.5 mM; 1 μl of Mg 2+ at 0.025 mol / L; 0.5 U of TAKARA rTaq; add ddH 2 O to make up to 10 μl. The PCR amplification program was: 95°C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec, for 35 cycles; 72°C for 5 min. 10% polyacrylamide gel electrophoresis was carried out for about 1 hour, followed by silver staining.

[0061] Through a single marker or a combination of multiple markers in Table 2, the allele type of the material at the HS10 locus can be determined for molecular marker-assisted selection. Of course, new polymorphic markers can also be obtained through sequencing or primer screening for allele type determination.

[0062] V. Breeding of α line and β line in "nuclear three-line system"

[0063] The progeny of the combination with 1892S have the advantages of strong heterosis, high yield, lodging resistance, heat resistance, etc. However, 1892S also has the significant disadvantages of high fertility conversion temperature and high risk of seed production. Previous studies found that 1892S is of the α allele type at the HS10 locus. If a fertile material with the same genetic background as 1892S and carrying the β allele type can be developed, the "nuclear three-line system" can be completed, and the problem of high fertility conversion temperature can be solved. For this purpose, using 1892F (fertile near-isogenic line of 1892S) as the recurrent parent and F1312 as the donor parent, male sterile plants were selected for continuous backcrossing until the 6th generation of backcrossing. Except for the fertility difference, there were no obvious differences in agronomic traits among the individual plants. At the same time, using 1892F as the recurrent parent, backcrossing with Guangzhan 63S, and selecting αR-type individual plants with the molecular marker chr10-13439993 for continuous backcrossing until the BC 6 generation with consistent agronomic traits of individual plants. Selecting sterile plants with the genotype of αβ as the female parent and fertile plants with the genotype of αR-type as the male parent for hybridization, and selecting βR-type fertile plants in the F 1 generation with the molecular marker chr10-13505990-1, self-crossing, planting the self-crossed offspring, and selecting fertile plants with the genotype of ββ type (fertile at both high and low temperatures, β line) by molecular marker-assisted selection, which is called 1892β, and RR-type individual plants are called 1892R. In a high-temperature environment, using 1892S (α line, 1892αS) as the female parent and 1892β line as the male parent for hybridization to produce 1892αβ line that is male sterile at both high and low temperatures.

[0064] 450S is a two-line male sterile line of the tms5 type. Through allelic verification, it was found that the allelic type at the HS10 locus is the α type. To develop 450S into a nuclear three-line system, 450S was crossed with 1892β, and 450S was used as the recurrent parent for backcrossing until the agronomic traits were stable after 4 generations of backcrossing. During the backcrossing process, molecular markers chr10-13505990-1 (the sequence is shown in Table 1) and tms5 functional markers (a functional marker for the rice thermosensitive male sterile gene tms5 and its application patent number: ZL 2015 1 0547897.4) were used to select single plants with the genotype αβ and heterozygous tms5 (genotype: αβTMS5tms5) for continued backcrossing. At the same time, using 450S as the recurrent parent and 1892R as the donor parent, hybridization and backcrossing were carried out for 4 generations, and molecular markers were used to select fertile single plants with the genotype αR and heterozygous tms5 (genotype: αR TMS5tms5). Using the αβTMS5tms5 type male sterile single plant as the female parent and the αR TMS5tms5 type fertile single plant as the male parent for hybridization, molecular markers were used to select fertile single plants of the βRTMS5TMS5 or βR TMS5tms5 type for selfing to save seeds. The selfed seeds were planted, and fertile single plants of the ββTMS5TMS5 type were selected to develop the 450β line. Using 450S as the female parent and 450β as the male parent for hybridization to produce the 450αβ line.

[0065] 601 is a fertile material of the R type. Using 601 as the recurrent parent, it was crossed with 1892S, and molecular markers of chr10-13505990-1 and tms5 were used to select fertile single plants of the αR TMS5tms5 type for continued backcrossing until the agronomic traits were stable after 5 generations of backcrossing. Then selfing was carried out, and molecular markers were used to select a two-line male sterile line of the ααtms5tms5 type, which is called the 601αS line. At the same time, using 601 as the recurrent parent and 1892β as the donor, molecular breeding was carried out to select βR type single plants for continued backcrossing for 5 generations, and then selfing was carried out, and molecular breeding was carried out to select ββ type single plants, which is called the 601β line. Using 601αS as the female parent and 601β as the male parent for hybridization to produce the 601αβ line.

[0066] 412 is a fertile material with the β type at the HS10 locus. Using 412 as the recurrent parent, it was crossed with 1892S, and molecular marker-assisted selection was used to select sterile single plants of the αβTMS5tms5 type for continued backcrossing for 6 generations. At the same time, using 412 as the recurrent parent, it was crossed with 1892R, and molecular marker-assisted selection was used to select sterile single plants of the βR type for continued backcrossing for 6 generations. Using the sterile single plant αβTMS5tms5 developed by high-generation backcrossing as the female parent and the βR type single plant as the male parent for hybridization, molecular markers were used to select αRTMS5tms5 single plants for selfing, and then two-line sterile plants of the ααtms5tms5 type were selected from the selfed offspring, that is, the 412αS line. Using 412αS as the female parent and 412 as the male parent to produce the 412αβ line.

[0067] The αS line shows male sterility under high-temperature conditions (such as in summer in the middle and lower reaches of the Yangtze River and the South China rice regions), and can be hybridized with the β line (fertile under both high and low temperatures) to produce the αβ line (male sterile under both high and low temperatures); under low-temperature conditions (such as in winter in Hainan), the αS line turns into male fertility and can reproduce normally. Conversely, by making the β line carry tms5 to breed the βS line and crossing it with the α line (fertile under both high and low temperatures), the production of the αβ line and the reproduction of the βS line can also be achieved.

[0068] VI. Breeding of the restorer line (R line) in the "nuclear three-line system"

[0069] For the selection of the restorer line, materials containing the R allele type at the HS10 locus can be selected through molecular markers as the restorer line, or directly test-crossed with the αβ line, and screened according to the seed-setting rate of the offspring. If the seed-setting rate of all individual plants is above 70%, this material can be used as the restorer line. It was found through test-crossing that the current main indica two-line and three-line restorer lines can all be used as the restorer line of the nuclear three-line system, such as Huazhan, Wushan Simiao, R0822, HK57, etc. They have a wide restorer spectrum, and there are no adverse performances in the offspring of the combinations, showing obvious heterosis.

[0070] VII. Technical route for the "nuclear three-line system"

[0071] 1) Creation of the heterozygous sterile line in the "nuclear three-line system"

[0072] First, determine the allele type of the rice (referred to as the target material) for which the heterozygous sterile line is to be bred at the HS10 locus. The target material is hybridized with the known α line and β line respectively, and the allele type is judged according to the fertility of the hybrid; if it is fertile with both α and β in the F1 generation, the target material is of the R type; if it is fertile with α and sterile with β, it is of the α type; if it is sterile with α and fertile with β, it is of the β type; if it is sterile or has poor fertility with both α and β, it is a new allele type or affected by other sterile loci.

[0073] Secondly, through primer screening and sequencing methods, polymorphic molecular markers of the target material and the known α, β, and R lines at the HS10 locus are screened for tracking the α, β, and R allele types during the breeding process. The molecular marker primers can preferably be the sequences shown in SEQ ID NO.1 - SEQ ID NO.10.

[0074] Thirdly, different breeding schemes are adopted according to the different allele types of the target material at the HS10 locus.

[0075] For the R-type target material, using this material as the recurrent parent, continuously backcross with the α-type and β-type materials respectively. Through molecular marker selection, select the plants with genotypes αR and βR respectively for continuous backcrossing until the agronomic traits of the high-generation backcross progeny are stable, and then self-cross. Molecular marker selection is used to obtain the near-isogenic line materials with genotypes αα and ββ. During the backcrossing process, the photoperiod- and thermo-sensitive genic male sterility genes can be selected simultaneously to develop the αS line or βS line. Cross the αS line with the β line or the βS line with the α line to produce the αβ-type heterozygous male sterile line. The specific process is shown in Figure 3 。

[0076] For the α-type target material, using this material as the recurrent parent, cross with the β-type and R-type materials respectively and then continuously backcross. During the crossing and backcrossing with the β line, the plants with genotype αβ are male sterile. Male sterile plants can be selected to continue backcrossing with the target material. At the same time, the target material is crossed with the R-type material, and molecular marker-assisted selection is used to select the plants with genotype αR for continuous backcrossing until the agronomic traits of the backcross progeny are stable. Subsequently, using the sterile αβ plant as the female parent and the fertile αR plant as the male parent for crossing, molecular markers are used to screen the plants with genotype βR, harvest the seeds, plant the F2 generation, and then use molecular markers to screen out the plants with genotype ββ. During the backcrossing process, the two-line male sterility genes can be introduced simultaneously to develop the αS line or βS line. The specific process is shown in Figure 4 。

[0077] Similarly, for the β-type material, cross with the α-type and R-type materials respectively and then continuously backcross, with molecular marker-assisted selection. Then, through the crossing and self-crossing of the sterile αβ line and the fertile βR line, develop the α line, and make one of the α line or β line carry the two-line male sterility gene to achieve the three-line matching. The specific process is shown in Figure 5 。

[0078] 2) Creation of the restorer line in the "nuclear three-line system"

[0079] If it is an R-allele type material, it can be directly used as the restorer line. If it is an α-type or β-type material, it can be crossed and continuously backcrossed with the R-type material, and molecular marker selection is used to replace the HS10 locus with the R allele to develop the restorer line; it can also be crossed and then self-crossed, and the R allele is selected from the self-crossed progeny to develop the restorer line. The seed setting rate of the progeny from the cross between the restorer line and the αβ line will be greater than 70%.

[0080] 3) Creation of the hybrid combination in the "nuclear three-line system"

[0081] The heterozygous sterile αβ line is crossed with the restorer line R to screen out the combinations with strong heterosis and produce hybrid seeds, ultimately achieving the matching of the "nuclear three-line system" and the utilization of heterosis.

[0082] In summary, by adopting the method for constructing the "nuclear three-line" breeding system of the present invention, it can be combined with other sterility systems (such as the two-line positive photo-thermo-sensitive or anti-photo-thermo-sensitive system) for large-scale production of heterozygous male sterile αβ lines. For materials that hope to utilize heterosis, the present invention can divide them into α type, β type or R type, and conduct cross-breeding and backcross-breeding with the other two types, and finally breed α lines, β lines and R lines to achieve the matching of the "nuclear three-line".

[0083] Although the principle of the present invention has been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are only explanations of the illustrative implementation manners of the present invention and do not limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention all fall within the protection scope of the present invention.

Claims

1. A method for establishing a "nuclear three-line" breeding system for rice, characterized in that, nearly isogenic line materials α line and β line carrying α allele type and β allele type at the HS10 locus are developed. Among them, the β line is a rice material that is fertile at both high and low temperatures, and the α line is a rice material that is sterile at both high and low temperatures after crossing with the β line. Under the condition that the sterile gene is in the sterile state, the α line is crossed with the β line to produce αβ type heterozygous sterile line seeds αβ, and then the heterozygous sterile line αβ is crossed with the R type restorer line to produce hybrid rice seeds. The R type restorer line is a rice material that restores fertility after crossing with the heterozygous sterile line αβ.

2. The method for establishing a "nuclear three-line" breeding system for rice according to claim 1, characterized in that: The HS10 locus refers to the interval of 13204998 - 13505990 on chromosome 10 of rice with 93-11 as the reference genome.

3. The method for establishing a "nuclear three-line" breeding system for rice according to claim 1, characterized in that: The β line is F1312 and its derivatives. The α line carrying the α allele type includes rice varieties 1892S, 1892F and their derivatives. The β line carrying the β allele type includes rice variety F1312 and its derivatives. The R line carrying the R allele type includes Guangzhan 63S, Huazhan, R0822, HK57 and their derivatives.

4. The method for establishing a "nuclear three-line" breeding system for rice according to claim 1, characterized in that: The Indel type molecular marker for detecting α, β and R allele types has primers with sequences as shown in SEQ ID NO.1 - SEQ ID NO.

10.

5. The method for establishing a "nuclear three-line" breeding system for rice according to claim 1, characterized in that: The sterile gene includes the sterile gene carried in the two-line positive photoperiod-temperature sensitive system or the reverse photoperiod-temperature sensitive system.

6. The method for establishing a "nuclear three-line" breeding system for rice according to claim 5, characterized in that: The sterile gene of the two-line positive photoperiod-temperature sensitive system includes tms5. The method includes introducing the tms5 gene into α line rice plants to develop the αS line, which is male sterile under high temperature conditions and turns male fertile under low temperature conditions. The αS line is crossed with the β line to produce the αβ line, which is male sterile at both high and low temperatures; or introducing tms5 into β line plants to develop the βS line, and crossing the βS line with the α line plants that are fertile at both high and low temperatures to produce the αβ line.

7. The method for establishing a "nuclear three-line" breeding system for rice according to claim 1, characterized in that, The allele types carried by the α line and the β line can be interchanged with each other.

8. A rice breeding method, characterized in that, judging the type of the target rice variety, determining the allele type of the target rice at the HS10 locus, and classifying the target rice variety into α type, β type or R type; respectively selecting the α line and the β line according to different allele types of the target rice at the HS10 locus: If the target rice is of the R type, using the R-type target rice as the recurrent parent, it is successively backcrossed with α- and β-type rice. Through molecular marker selection, individual plants with genotypes αR and βR are selected for continuous backcrossing until the agronomic traits of the high-generation are stable. Then, self-crossing is carried out, and near-isogenic line materials with genotypes αα and ββ are selected by molecular markers, and finally, the α line and β line with the target rice as the genetic background are developed; If the target rice is of the α type, using the α-type target rice as the recurrent parent, it is successively backcrossed after being hybridized with β-type and R-type rice. During the hybridization and backcrossing with the β line, individual plants with the genotype αβ are male sterile. Select male sterile individual plants to continue backcrossing with the target material. At the same time, the target material is hybridized with the R line, and molecular marker-assisted selection is used to select individual plants with the genotype αR for continuous backcrossing until the agronomic traits of the backcross offspring are stable. Subsequently, using the sterile αβ individual plant as the female parent and the fertile αR individual plant as the male parent for hybridization, molecular markers are used to screen individual plants with the genotype βR, the seeds are harvested, the F2 is planted, and then individual plants with the genotype ββ are screened out by molecular markers, and finally, the β line with the target rice as the background is developed; Similarly, if the target rice is of the β type, the β-type target rice is successively backcrossed after being hybridized with the α line and the R line, molecular marker-assisted selection is used, and then through the hybridization and self-crossing of the sterile αβ line and the fertile βR line, the α line with the target rice as the background is developed.

9. A breeding method for the restorer line in a "nuclear three-line" breeding system Characterized in that Using molecular markers to select rice with the HS10 locus being of the R type as the restorer line; or selecting rice with a seed setting rate of more than 70% in the offspring of the hybridization with the αβ line as the restorer line.

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