Breeding method of photo-thermo-sensitive genic male sterile line of long-grain fragrant disease-resistant rice

Abstract

The invention relates to the technical field of rice breeding, in particular to a breeding method of a photo-thermo-sensitive genic male sterile line of long-grain fragrant disease-resistant rice. The method comprises the following steps: hybridizing a male parent and a female parent of rice to obtain F1 seeds, planting the F1 seeds, detecting genes by using molecular markers, selecting single plants carrying positive hybrid genes, cutting stumps, regenerating, treating at 19-21 DEG C until the plants are normal and fertile to obtain F2 seeds, planting an F2 group, and combining a growth period and phenotypes to obtain the rice group. Selecting a single plant which carries a target gene, has a growth period close to that of the female parent, is good in fruit setting and has good appearance quality after rice stripping inspection to obtain F3 seeds, and finally breeding the long-grain fragrant disease-resistant rice photo-thermo-sensitive genic male sterile line, so as to obtain the long-grain fragrant disease-resistant rice photo-thermo-sensitive genic male sterile line. The breeding method provided by the invention is short in breeding period and relatively high in breeding efficiency, and can improve multiple characters at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of rice breeding, in particular to a method for breeding a photothermosensitive genic male sterile line of long-grain fragrant disease-resistant rice. Background Art

[0002] Rice is one of the most important food crops in my country and even in the world. Under the conditions of a growing population and limited arable land, the innovation of rice breeding technology has played an indispensable role in further increasing rice production and ensuring national grain self-sufficiency. Among them, the original invention of two-line hybrid rice is another major technological leap after dwarf breeding and three-line hybrid rice. Two-line hybrid rice has gradually become the dominant type of rice production due to its advantages such as simplified seed production procedures, free combination, and no negative cytoplasmic effects.

[0003] As people's living standards improve, they pay more and more attention to the quality and nutritional health of rice. Therefore, rice varieties that are high-yield, high-quality (good taste, fragrance, etc.), and multi-resistant are becoming more and more popular in the market. Among them, long-grain fragrant rice is favored by consumers, but the number of fragrant two-line hybrid rice varieties that have been cultivated is relatively small, and the promotion area is limited, which cannot meet the urgent demand for fragrant rice in the existing consumer market. Creating fragrant two-line sterile lines is an important way to cultivate fragrant two-line hybrid rice. At present, breeders mainly use conventional hybrid breeding strategies combined with phenotypic identification to achieve multi-trait selection, but the breeding population is large, the breeding cycle is long, and the breeding efficiency is low.

[0004] Photoperiod-temperature-sensitive nuclear sterile lines are the core of two-line hybrid rice, and their main breeding methods include screening of naturally variable sterile plants of existing varieties (such as Nongken 58S and Annong S-1), mutation breeding (such as 5460S) and hybrid breeding strategies. Among them, hybrid breeding is the most commonly used method at present. The specific process is: select a sterile line with obvious photoperiod-temperature sensitivity, and the critical light length and critical temperature for fertility conversion meet the local ecological conditions, and hybridize it with the target variety; plant F1 plants and harvest F2 seeds; plant the F2 segregating population under long light and high temperature conditions, and select (excellent) sterile plants at heading; transplant the selected individual plants to short light and low temperature conditions for regeneration, and when the regenerated plants head, bag the rice ears of the fertile plants and harvest the self-pollinated seeds; plant F3 strains under appropriate light and temperature conditions, select individual plants with different fertility conversion conditions according to the breeding goals, and harvest the self-pollinated seeds; for example, to If the sterile lines with a critical light length of 14h and a critical temperature of 23.5℃ are selected for fertility conversion, they must be planted under such conditions, sterile plants must be selected, and seeds must be harvested for regeneration and reproduction; F4-F6 can be planted under fertile conditions, and other agronomic traits and quality can be selected, and their heredity can be stabilized; the F7 generation is identified for fertility conversion characteristics of different light and temperature combinations in an artificial climate chamber, and the critical temperature and critical light length of fertility conversion of the selected sterile lines are determined. At the same time, the fertility dynamics under different ecological conditions are observed, and the ecological areas where the sterile lines may be applied are evaluated, combined testing and matching, general combining ability analysis, etc., are finally selected for practical light-temperature sensitive nuclear sterile lines. However, traditional breeding methods have the disadvantages of high uncertainty in breeding target selection (low selection efficiency, etc.), difficulty in stabilizing the genome, and long breeding cycles.

[0005] At present, there have been relevant reports on the research of rice photothermosensitive genic male sterile line breeding methods. For example, the invention patent with Chinese patent authorization announcement number CN103598085B and authorization announcement date September 23, 2015 provides a method for breeding rice photothermosensitive genic male sterile lines, which is characterized by: selecting high-quality, rice blast-resistant, high-combining ability, early-maturing medium-length indica photothermosensitive genic male sterile line "Guangzhan 63S" as the female parent, selecting a compact plant type with good lodging resistance, high quality, rice blast resistance, and a deposit number of CGMCCN bred by the Jiangsu Coastal Agricultural Science Research Institute The conventional mid-season indica rice intermediate material "Yandao 229" of O.7619 was used as the male parent, and the hybridization and generation of F1 seeds were harvested. From F2-F6 generations, each generation selected excellent individual plants with good photothermosensitive nuclear sterility characteristics, excellent quality and compact plant type. Finally, the mid-season indica photothermosensitive nuclear sterile line Yan 166S with compact plant type, high quality, resistance to rice blast and early maturity was bred. Although this sterile line has better solved the shortcoming of poor lodging resistance of the new two-line hybrid rice combination prepared with the indica photothermosensitive nuclear sterile line Guangzhan 63S. However, during the breeding process, each generation needs to be identified for light and temperature fertility, and a large selection population is required. The single phenotypic selection is not only easily affected by environmental and human factors, but also has a low efficiency in selecting multiple breeding targets at the same time. In addition, the invention patent with Chinese patent publication number CN105918110A, a method for cultivating a fragrant high-quality rice sterile line, and the invention patent with Chinese patent authorization announcement number CN101889544B both mention the use of anther in vitro culture technology (abbreviated as "flower culture") to flower culture the microspores of the re-cross F1, and then conduct phenotypic and fertility identification on the flower culture offspring. Compared with traditional breeding, it has an absolute advantage in the breeding cycle and can quickly achieve homozygosity and stability. However, the use of re-cross F1 (or F1) for flower culture has high requirements on the flower culture offspring population. A sufficiently large variation population (for example, 1,000 mature seedlings) is required to select individual plants that meet the breeding goals, which greatly increases the workload of flower culture.

[0006] In view of the defects and problems existing in the prior art such as difficulty in achieving multiple goals, long breeding cycle and low breeding efficiency, the present invention provides a method for breeding a photo-thermo-sensitive nuclear sterile line of long-grain fragrant disease-resistant rice with a short breeding cycle, high breeding efficiency and simultaneous improvement of multiple traits. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for breeding a photothermosensitive genic male sterile line of long-grain fragrant disease-resistant rice. The breeding method provided by the present invention has a short breeding cycle, high breeding efficiency and can improve multiple traits at the same time.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for breeding a photothermosensitive genic male sterile line of long-grain fragrant disease-resistant rice, comprising the following steps:

[0010] 1) hybridizing the male and female parents of rice to obtain F1 seeds;

[0011] The male parent carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene;

[0012] The female parent carries a recessive homozygous rice blast resistance gene, a recessive homozygous rice grain gene, a positive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene;

[0013] 2) Planting the F1 seeds obtained in step 1), detecting the rice blast resistance gene and the rice aroma gene using molecular markers, selecting individual plants carrying positive heterozygous genes, cutting the stumps for regeneration, and treating at 19-21° C. until they are normally fertile, to obtain F2 seeds;

[0014] 3) Planting the F2 seeds obtained in step 2), and selecting individual plants carrying the target gene, with a growth period biased towards the female parent, good fruit set and good rice grain appearance quality according to the growth period and phenotype, to obtain F3 seeds;

[0015] The target genes are rice aroma gene, rice blast resistance gene and rice grain gene;

[0016] The rice fragrance gene is recessive homozygous, the rice blast resistance gene is positive homozygous or positive heterozygous, and the rice grain gene is positive homozygous or positive heterozygous;

[0017] 4) Planting the F3 seeds obtained in step 3), selecting individual plants with homozygous target genes based on agronomic traits and flowering habits, cutting the stumps for regeneration, and dividing the obtained rice stumps into two parts. During the fertility sensitive period, one part is subjected to fertility conversion critical temperature screening in an artificial climate box with an average daily temperature of 23° C. and a sunshine duration of 14 h, and the other part is treated in a cold water pool at 19-21° C. until it is normally fertile. Individual plants that are identified as completely sterile in the artificial climate box with a pollen sterility of ≥99.5% and are normally fruitful after treatment in the cold water pool are selected to obtain F4 seeds;

[0018] 5) Planting the F4 seeds obtained in step 4), selecting strains based on the target gene, fertile population consistency, growth period, agronomic traits, rice appearance and fruit setting rate, and culturing in vitro the anthers of the strains at the pollen mononucleus edge stage of the panicle stage to obtain H0 generation anther culture strains;

[0019] 6) The H0 generation flower culture strain obtained in step 5) is combined with gene detection, screening and identification of the critical temperature for fertility conversion in an artificial climate box with an average daily temperature of 23° C. and a sunshine duration of 14 h, agronomic traits and flowering habits to breed a photothermosensitive genic male sterile line of long-grain fragrant rice with qualified fertility and disease resistance.

[0020] Preferably, in step 1), the male parent is MS411 and the female parent is Longke 638S.

[0021] Preferably, the F1 seeds in step 1) carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain quality gene, a positive heterozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

[0022] Preferably, the molecular markers in step 2) are RM5766 and Aro, the molecular marker RM5766 detects the rice blast resistance gene, and the molecular marker Aro detects the rice aroma gene.

[0023] Preferably, the F2 seeds in step 2) carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain quality gene, a positive heterozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

[0024] Preferably, the F3 seeds in step 3) carry a positive heterozygous or positive homozygous rice blast resistance gene, a positive heterozygous or positive homozygous rice grain quality gene, a recessive homozygous rice aroma gene, and a recessive homozygous rice thermo-sensitive sterility gene.

[0025] Preferably, the target genes in step 4) are rice blast resistance genes, rice grain quality genes and rice aroma genes.

[0026] Preferably, the F4 seeds in step 4) carry a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice thermo-sensitive sterility gene.

[0027] Preferably, the target genes in step 5) are rice blast resistance genes, rice grain quality genes and rice aroma genes;

[0028] The H0 generation flower culture strain carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice fragrance gene and a recessive homozygous rice thermosensitive sterility gene.

[0029] Preferably, in step 6), the long-grain fragrant disease-resistant rice photothermosensitive genic male sterile line carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice fragrance gene and a recessive homozygous rice thermosensitive male sterile gene.

[0030] In the present invention, the rice blast resistance gene is referred to as Pi1 gene, the rice grain gene is referred to as GL7 gene, the rice fragrance gene is referred to as fgr gene, and the rice thermosensitive sterility gene is referred to as tms5 gene.

[0031] Beneficial effects of the present invention:

[0032] 1. Molecular marker-assisted selection technology is used to improve multiple traits. In the cultivation of a long-grain fragrant disease-resistant rice photothermosensitive nuclear sterile line of the present invention, the molecular marker-assisted selection technology can be used to detect the main effect target gene from generation to generation, avoiding the influence of environmental factors, and can simultaneously carry out multi-target trait selection, greatly improving the selection efficiency. Conventional hybridization or multiple cross-type aggregate breeding (such as the patent with publication number CN110050685A) relies only on phenotypic selection, which makes it difficult to achieve early generation selection, and the phenotype is easily affected by the environment. There are often shortcomings such as long breeding cycle and difficult to achieve breeding goals; in addition, the improvement of sterile lines combined with molecular marker-assisted selection technology often only improves a single trait, such as rice blast resistance (patent authorization announcement number: CN108496790B), which cannot meet the current demand for simultaneous selection of multiple traits.

[0033] 2. On the basis of early generation selection, low-cost anther breeding is used to quickly achieve the goal of trait improvement. Since the present invention starts fertility screening from the F4 generation where the population traits are basically stable, it is possible to combine anther breeding with relatively small anther breeding offspring on the basis of target gene detection and agronomic trait selection in the F2 and F3 generations to achieve the effect of target trait selection and rapid homozygosity of the strain, greatly reducing the demand for the size of the anther breeding population and quickly selecting stable strains. In existing anther breeding cases (such as authorization announcement number: CN101889544B), in vitro culture is generally carried out using F1 or recrossed F1 anthers, and a sufficiently large selection population (equivalent to the F2 population of conventional breeding) must be used to achieve the breeding goal. In the case of relatively low conversion efficiency of indica rice, the workload and investment cost of anther breeding are increased, and a relatively small anther breeding population can often only quickly stabilize traits, but it is difficult to achieve the selection goal.

[0034] 3. Use an artificial climate box to carry out flower culture on individual plants after fertility screening of the F4 line, significantly improving the efficiency of fertility selection of two-line sterile lines. Since the present invention first carries out artificial climate box fertility identification in the F4 generation, and carries out flower culture on the offspring of individual plants (23°C treatment, pollen sterility ≥99.5%) screened based on low temperature selection pressure, and carries out a second artificial climate box fertility identification on the flower culture line H1, it is possible to obtain a new sterile line with stable fertility and low critical temperature for fertility conversion (≤23°C) with a greater probability, ensuring the dual effects of main agronomic trait selection and light temperature fertility identification. There are existing flower culture cases (such as authorization announcement number: CN101889544B), all of which carry out a fertility identification on the stable flower culture line, and the proportion of individual plants that meet the fertility requirements is low, while the proportion of lines with excellent agronomic traits is even lower, with high randomness and blindness.

[0035] Other notes:

[0036] 1) The target trait genes such as the rice blast resistance gene Pi1 and the grain shape gene GL7 described in the present invention can be replaced by cloned major effect genes (such as Pi2, Pigm, etc.; GS3, GL3, etc.) according to the requirements of rice blast resistance or grain shape in different rice ecological zones to achieve similar breeding purposes;

[0037] 2) The size of the selection population described in the present invention is determined according to the selection intensity of the target gene and the selection intensity of the main agronomic traits, and can be adjusted according to actual needs;

[0038] 3) The fertility of the photothermosensitive genic male sterile line involved in the present invention is mainly regulated by temperature. Within a certain temperature range, it is fertile at low temperature and sterile at high temperature. Therefore, under high temperature in summer, the selected sterile individual plants need to be cut and regenerated and then treated with a certain low temperature to change from sterile to fertile and harvest seeds, while under low temperature in winter, they can often bear fruit naturally. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0040] Figure 1 The molecular detection results of the target genes Pi1, fgr and GL7 for the newly bred 6 sterile lines;

[0041] Figure 2 The grain type performance of 6 newly bred sterile lines;

[0042] Figure 3 Flow chart of the breeding method of the present invention, wherein F1, F2, F3 and F4 represent breeding generations (first to fourth generations), H0, H1 and H2 represent the first, second and third generations of flower culture lines respectively; Pi1 is a dominant rice blast resistance gene, GL7 is a dominant rice grain type gene, fgr is a recessive rice fragrance gene, tms5 is a recessive rice thermosensitive sterility gene; the symbols "Pi1Pi1", "Pi1pi1" and "pi1pi1" represent the three genotypes of Pi1 gene positive homozygous, positive heterozygous and negative homozygous, respectively, and other gene symbols can be deduced by analogy; "MAS" is Molecular Marker-assisted The abbreviation of Selection, namely "molecular marker-assisted selection", is a GSR40K chip (authorized patent number CN111684113A) developed by Wuhan Shuanglvyuan Chuangxin Technology Research Institute Co., Ltd. It uses the international cutting-edge functional genomics research results to mine the massive whole genome sequence of rice, screen out representative and accurately detectable SNP markers, design them into probes, and add functional markers closely related to disease resistance, insect resistance, drought resistance, high fertilizer efficiency, high quality, and high yield. It is developed using the leading Illumina Infinium chip manufacturing technology. DETAILED DESCRIPTION

[0043] The present invention provides a method for breeding a photothermosensitive genic male sterile line of long-grain fragrant disease-resistant rice, comprising the following steps:

[0044] 1) hybridizing the male and female parents of rice to obtain F1 seeds;

[0045] The male parent carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene;

[0046] The female parent carries a recessive homozygous rice blast resistance gene, a recessive homozygous rice grain gene, a positive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene;

[0047] 2) Planting the F1 seeds obtained in step 1), detecting the rice blast resistance gene and the rice aroma gene using molecular markers, selecting individual plants carrying positive heterozygous genes, cutting the stumps for regeneration, and treating at 19-21° C. until they are normally fertile, to obtain F2 seeds;

[0048] 3) Planting the F2 seeds obtained in step 2), and selecting individual plants carrying the target gene, with a growth period biased towards the female parent, good fruit set and good rice grain appearance quality according to the growth period and phenotype, to obtain F3 seeds;

[0049] The target genes are rice aroma gene, rice blast resistance gene and rice grain gene;

[0050] The rice fragrance gene is recessive homozygous, the rice blast resistance gene is positive homozygous or positive heterozygous, and the rice grain gene is positive homozygous or positive heterozygous;

[0051] 4) Planting the F3 seeds obtained in step 3), selecting individual plants with homozygous target genes based on agronomic traits and flowering habits, cutting the stumps for regeneration, and dividing the obtained rice stumps into two parts. During the fertility sensitive period, one part is subjected to fertility conversion critical temperature screening in an artificial climate box with an average daily temperature of 23° C. and a sunshine duration of 14 h, and the other part is treated in a cold water pool at 19-21° C. until it is normally fertile. Individual plants that are identified as completely sterile in the artificial climate box with a pollen sterility of ≥99.5% and are normally fruitful after treatment in the cold water pool are selected to obtain F4 seeds;

[0052] 5) Planting the F4 seeds obtained in step 4), selecting strains based on the target gene, fertile population consistency, growth period, agronomic traits, rice appearance and fruit setting rate, and culturing in vitro the anthers of the strains at the pollen mononucleus edge stage of the panicle stage to obtain H0 generation anther culture strains;

[0053] 6) The H0 generation flower culture strain obtained in step 5) is combined with gene detection, an artificial climate with an average daily temperature of 23° C. and a sunshine duration of 14 h to screen and identify the critical temperature for fertility conversion, agronomic traits and flowering habits, and a long-grain fragrant disease-resistant rice photothermosensitive genic male sterile line is selected.

[0054] The present invention hybridizes the male parent and the female parent of rice to obtain F1 seeds; the male parent carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain quality gene, a positive homozygous rice fragrance gene and a recessive homozygous rice thermosensitive sterility gene; the female parent carries a negative homozygous rice blast resistance gene, a negative homozygous rice grain quality gene, a negative homozygous rice fragrance gene and a recessive homozygous rice thermosensitive sterility gene.

[0055] In the present invention, the male parent is preferably MS411, and the female parent is preferably Longke 638S. In the present invention, the MS411 is a photosensitive genic male sterile line with a short growth period, long grain and fragrance, and resistance to rice blast, bred by the Food Crop Research Institute of the Hubei Academy of Agricultural Sciences; Longke 638S is an excellent photosensitive genic male sterile line with strong general combining ability and a relatively long growth period, bred by the Hunan Yahua Seed Science Research Institute. More than 90 hybrid rice varieties have been selected and bred and passed provincial or national approval, with an annual promotion area of ​​more than 1 million hectares. In the present invention, the F1 seeds preferably carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain gene, a positive heterozygous rice fragrance gene, and a recessive homozygous rice thermosensitive sterile gene.

[0056] The F1 seeds obtained by planting the present invention are used to detect the rice blast resistance gene and the rice aroma gene by molecular markers, and single plants carrying positive heterozygous genes are selected, and the stumps are cut and regenerated, and the seeds are treated at 19-21° C. until they are normally fertile to obtain F2 seeds. In the present invention, the molecular markers are preferably RM5766 and Aro (1) Chen Zhiwei, Guan Huazhong, Wu Weiren, et al. Screening and application of SSR markers linked to the rice blast resistance gene Pi-1 [J]. Journal of Fujian Agricultural University, 2005 (1): 74-77. 2) Du Xueshu, Xia Mingyuan, Li Jinbo, et al. Molecular marker-assisted selection for breeding of aromatic rice restorer lines [J]. Journal of Huazhong Agricultural University, 2009, 28 (6): 651-654.), the molecular marker RM5766 is preferably used to detect the rice blast resistance gene, and the molecular marker Aro is preferably used to detect the rice aroma gene. The present invention does not specifically limit the detection method, and those skilled in the art can use conventional methods. In the present invention, the F2 seeds preferably carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain gene, a positive heterozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

[0057] The F2 seeds obtained by planting the present invention are selected, in combination with the growth period and phenotype, to carry the target gene, the growth period is biased towards the maternal parent, the fruiting rate is good (fruiting rate is 50%-80%) and the rice peeling inspection appearance quality is good (mainly refers to the slender grain shape, low chalkiness and good transparency) to obtain F3 seeds; the target gene is a rice fragrance gene, a rice blast resistance gene and a rice grain gene; the rice fragrance gene is recessive homozygous, the rice blast resistance gene is positive homozygous or positive heterozygous, and the rice grain gene is positive homozygous or positive heterozygous. The present invention preferably detects the rice blast resistance gene, the rice fragrance gene and the rice thermosensitive sterility gene by molecular markers. In the present invention, the molecular markers are preferably RM5766 and Aro, the molecular marker RM5766 preferably detects the rice blast resistance gene, and the molecular marker Aro preferably detects the rice fragrance gene. The present invention does not specifically limit the detection method, and those skilled in the art can use conventional methods. In the present invention, the F3 seeds preferably carry a positive heterozygous or positive homozygous rice blast resistance gene, a positive heterozygous or positive homozygous rice grain gene, a recessive homozygous rice aroma gene, and a recessive homozygous rice thermosensitive sterility gene.

[0058] The F3 seeds obtained by planting the present invention are selected based on agronomic traits and flowering habits, and single plants with homozygous target genes are selected, and the stumps are cut and regenerated. The obtained rice stumps are divided into two parts. During the fertility sensitive period, one part is screened for fertility conversion critical temperature in an artificial climate box with a daily average temperature of 23°C and a sunshine duration of 14h, and the other part is treated in a cold water pool of 19-21°C until it is normally fertile. The single plants identified as completely sterile (pollen sterility ≥ 99.5%) by the artificial climate box and normal fruiting by the cold water pool treatment are selected to obtain F4 seeds. In the present invention, the target genes are rice blast resistance genes, rice grain genes and rice fragrance genes. The method for detecting the target genes of the present invention is the same as above, and will not be repeated here. In the present invention, the F4 seeds preferably carry positive homozygous rice blast resistance genes, positive homozygous rice grain genes, recessive homozygous rice fragrance genes and recessive homozygous rice temperature-sensitive sterility genes. In the present invention, the fertility sensitive period is preferably the spikelet differentiation stage III-IV.

[0059] The F4 seeds obtained by planting the present invention are selected to obtain strains in combination with the target gene, fertile period population consistency, growth period, agronomic traits, rice appearance and fruit setting rate, and the anthers of the single plant panicle stage of the strain that are in the pollen mononucleus side stage are cultured in vitro to obtain the H0 generation flower culture strain. In the present invention. The target gene is preferably a rice blast resistance gene, a rice grain gene and a rice aroma gene. The method for detecting the target gene of the present invention is the same as above and will not be repeated here. In the present invention, the H0 generation flower culture strain preferably carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice temperature-sensitive sterility gene.

[0060] The present invention combines the obtained H0 generation flower culture strains with gene detection, identification of 14 hours per day at 23°C, agronomic traits and flowering habits to breed long-grain fragrant disease-resistant rice light-temperature sensitive genic male sterile lines. In the present invention, the long-grain fragrant disease-resistant rice light-temperature sensitive genic male sterile lines preferably carry a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice fragrance gene and a recessive homozygous rice temperature-sensitive sterile gene.

[0061] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] 1. Implementation Plan

[0064] 1) Parents

[0065] MS411 is a photosensitive nuclear male sterile line with a short growing period, long grains and fragrant flavor and resistance to rice blast bred by the Grain Crops Research Institute of Hubei Academy of Agricultural Sciences; Longke 638S is an excellent photosensitive nuclear male sterile line with strong general combining ability and a relatively long growing period bred by Hunan Yahua Seed Science Research Institute. More than 90 hybrid rice varieties have been selected and bred and passed provincial or national approval, with an annual promotion area of ​​more than 1 million hectares.

[0066] 2) Breeding process

[0067] In the winter of 2020, a hybrid was conducted in Hainan using Longke 638S as the female parent and MS411 as the male parent to obtain F1 seeds;

[0068] In the summer of 2021, 20 F1 seedlings were planted in Wuhan. The rice blast resistance gene Pi1 and the fragrance gene fgr were detected by molecular markers RM5766 and Aro, respectively, through PCR amplification and polyacrylamide gel electrophoresis. Double-gene heterozygous plants were selected, and the stumps were cut and regenerated. They were treated at low temperature (19℃-21℃, the same below) in an artificial temperature-controlled cold water pool until they were normal and fertile, and sufficient F2 seeds were harvested;

[0069] In the winter of 2021, 1,200 F2 populations were planted in Hainan. Molecular markers RM5766, Aro, and InDel marker M-SGL7 were used to molecularly detect Pi1, fgr, and grain shape gene GL7, respectively. Combined with growth period and phenotypic selection, 24 excellent individual plants were selected that carried the three target genes at the same time (homozygous for fgr, homozygous or heterozygous for Pi1 or GL7), had a growth period biased towards Longke 638S, had a fruiting rate higher than 50%, and had slender rice grains, low chalkiness, and good transparency in rice peeling inspection. F3 family seeds were harvested;

[0070] In the summer of 2022, 100 plants of each of the 24 F3 families were planted in Wuhan, and molecular tests were performed on Pi1, fgr, and GL7. Combining the main agronomic traits such as plant height, growth period, number of effective panicles, flowering habit traits such as stigma exposure rate, and molecular test results, 5 families with a total of 40 homozygous single plants of the target genes were selected, and the stumps were cut and regenerated. The rice stumps were divided into two, and when they grew to the III-IV stage of panicle differentiation (i.e., the fertility sensitive period), one of the rice stumps was subjected to fertility identification in an artificial climate box at 23℃ / 14h, and the fertility performance of each single plant was recorded. The other corresponding rice stump was treated in an artificial temperature-controlled cold water pool, and F4 seeds were harvested;

[0071] In the winter of 2022, Hainan will plant 40 F4 lines in three phases (November 10, November 20, and November 30), take mixed samples at the seedling stage, and use molecular detection of Pi1, fgr, and GL7 to verify the target genes. Combined with phenotypes such as fertile population consistency, growth period, main agronomic traits, rice appearance, and fruit setting rate, 18 excellent lines will be selected. The lines corresponding to the second or third phase (depending on the specific growth period) will select anthers with single panicles in the pollen mononucleate marginal stage, and carry out anther in vitro culture. After anther culture, stable H0 generation anther culture lines will be obtained;

[0072] In the summer of 2023, 18 H0 flower culture lines will be transplanted in Wuhan to carry out target gene detection and verification, artificial climate chamber fertility identification, main agronomic traits and flowering habit investigation, etc., and 6 new sterile lines with good flowering habits and low critical temperature for fertility conversion will be selected. The fertile single plants will be cut and regenerated, and treated with low temperature and cold water until they are fertile and harvested H1 seeds;

[0073] In the winter of 2023, the H1 strain will be sown in Hainan in three phases (November 20, November 30 and December 10), with 80 plants planted in each phase. The main agronomic traits including 1000-grain weight and fruit setting rate will be investigated. In addition, 6 sterile strains will be isolated and propagated in the plot, and the target genes Pi1, fgr and GL7 will be detected and verified by molecular markers. The GSR40K chip will be used for functional gene detection and genetic background analysis, and the quality of mixed grain samples will be analyzed. Sufficient H2 strain seeds will be harvested. Later, the 6 sterile lines will be numbered YS01-YS06, which is the new fragrant long-grain disease-resistant light-temperature sensitive nuclear sterile line.

[0074] 2. Main agronomic traits of the new sterile lines

[0075] 1) Effect of molecular marker-assisted selection

[0076] Through molecular marker-assisted selection, the rice blast gene Pi1, aroma gene fgr and grain shape gene GL7 were successfully introduced from MS411 into Longke 638S. Combined with phenotypic selection, six new sterile lines with long grains and aroma were developed, YS01-YS06. All six new sterile lines carry homozygous Pi1, fgr and GL7 genes ( Figure 1 ).

[0077] 2) Main agronomic traits of the new sterile lines during the sterile and fertile periods

[0078] The sowing-starting period of the Wuhan sterile period of the six new sterile lines was 79.3d-81.7d, which was 3.6d-6.0d longer than that of the control MS411, and the maturity period was significantly improved; the plant height was 80.7cm-90.7cm, which was between MS411 and Longke 638S, and significantly higher than MS411; the number of effective ears, average ear length, and number of spikelets per ear of YS01, YS02 and YS04 were all increased compared with MS411; the stigma exposure rate of the six new sterile lines was 55.8%-71.8%, among which YS04 had the highest stigma exposure rate; after aroma identification, Longke 638S had no aroma, and the new sterile lines and the control MS411 were both identified as having aroma, which was consistent with the results of molecular detection.

[0079] Table 1 Main agronomic traits of new long-grain fragrant disease-resistant photothermosensitive genic male sterile lines (Wuhan sterile period)

[0080]

[0081]

[0082] The fertile period from sowing to harvest in Hainan of the six new sterile lines was 79.5-83.5 days, 4.5-7.0 days longer than that of MS411; the plant height was 67.5-74.6 cm, and the average ear length was 20.9-21.8 cm, 0.3-2.1 cm longer than that of MS411; the total number of grains per ear was 107.6-161.2, 7.6-61.2 more than that of the control; the fruit setting rate was 51.9-76.2%, and the fruit setting rates of YS04, YS05, and YS06 were all higher than those of MS411, and the reproductive yield was high; the single plant weight of the new lines was 14.5-22.0 g, 0.1-7.6 g heavier than the control, among which YS03 had the highest fruit setting rate and single plant weight.

[0083] Table 2 Main agronomic traits of new long-grain fragrant disease-resistant photothermosensitive genic male sterile lines (fertile period in Hainan)

[0084]

[0085] 3) Quality performance of new sterile rice lines

[0086] The results of rice quality inspection and analysis showed (Table 3) that the polished rice rate of the six new sterile lines was 56.1-63.1%, the grain length was 7.0-7.5 mm, and the length-width ratio was 3.7-4.1. All of them were long grains. The introduction of grain shape genes significantly improved the grain shape ( Figure 2 ); Among them, the rice quality of YS02 was poor, with a head rice rate of 56.1%, 8.5% lower than that of MS411, a chalky grain rate and chalkiness of 26.0% and 5.1% higher than that of the control, respectively, and a low alkali digestion value of 3.0, similar to Longke 638S. Among the new sterile lines, YS06 had a head rice rate of 62.5%, a grain length of 7.4 mm, a length-to-width ratio of 4.1, a chalky grain rate of 20.0%, a chalkiness of 3.5%, and an alkali digestion value of 6.6. All rice quality data were most similar to those of MS411.

[0087] Table 3 Quality traits of new long-grain fragrant disease-resistant photothermosensitive genic male sterile rice lines

[0088]

[0089] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for breeding long-grain fragrant disease-resistant rice photothermosensitive genic male sterile line, characterized in that: The following steps are involved: 1) hybridizing the male and female parents of rice to obtain F1 seeds; The male parent carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene; The female parent carries a recessive homozygous rice blast resistance gene, a recessive homozygous rice grain gene, a positive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene; 2) Planting the F1 seeds obtained in step 1), detecting the rice blast resistance gene and the rice aroma gene using molecular markers, selecting individual plants carrying positive heterozygous genes, cutting the stumps for regeneration, and treating at 19-21° C. until they are normally fertile, to obtain F2 seeds; 3) Planting the F2 seeds obtained in step 2), and selecting individual plants carrying the target gene, with a growth period biased towards the female parent, good fruit set and good rice grain appearance quality according to the growth period and phenotype, to obtain F3 seeds; The target genes are rice aroma gene, rice blast resistance gene and rice grain gene; The rice fragrance gene is recessive homozygous, the rice blast resistance gene is positive homozygous or positive heterozygous, and the rice grain gene is positive homozygous or positive heterozygous; 4) Planting the F3 seeds obtained in step 3), selecting individual plants with homozygous target genes based on agronomic traits and flowering habits, cutting the stumps for regeneration, and dividing the obtained rice stumps into two parts. During the fertility sensitive period, one part is subjected to fertility conversion critical temperature screening in an artificial climate box with an average daily temperature of 23° C. and a sunshine duration of 14 h, and the other part is treated in a cold water pool at 19-21° C. until it is normally fertile, and selecting individual plants that are completely sterile identified in the artificial climate box, with a pollen sterility of ≥99.5%, and are normally fruited after treatment in the cold water pool, to obtain F4 seeds; 5) Planting the F4 seeds obtained in step 4), selecting strains based on the target gene, fertile population consistency, growth period, agronomic traits, rice appearance and fruit setting rate, and culturing in vitro the anthers of the strains at the pollen mononucleus edge stage of the panicle stage to obtain H0 generation anther culture strains; 6) The H0 generation flower culture strain obtained in step 5) is combined with gene detection, identification of treatment at 23° C. for 14 hours per day, agronomic traits and flowering habits to select a long-grain fragrant disease-resistant rice photothermosensitive genic male sterile line.

2. The breeding method according to claim 1, characterized in that: In step 1), the male parent is MS411 and the female parent is Longke 638S.

3. The breeding method according to claim 1, characterized in that: In the step 1), the F1 seeds carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain quality gene, a positive heterozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

4. The breeding method according to claim 1, characterized in that: The molecular markers in step 2) are RM5766 and Aro, wherein the molecular marker RM5766 detects the rice blast resistance gene, and the molecular marker Aro detects the rice aroma gene.

5. The breeding method according to claim 1, characterized in that: In the step 2), the F2 seeds carry a positive heterozygous rice blast resistance gene, a positive heterozygous rice grain quality gene, a positive heterozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

6. The breeding method according to claim 1, characterized in that: In the step 3), the F3 seeds carry a positive heterozygous or positive homozygous rice blast resistance gene, a positive heterozygous or positive homozygous rice grain gene, a recessive homozygous rice aroma gene, and a recessive homozygous rice thermosensitive sterility gene.

7. The breeding method according to claim 1, characterized in that: The target genes in step 4) are rice blast resistance genes, rice grain quality genes and rice aroma genes.

8. The breeding method according to claim 1, characterized in that: In the step 4), the F4 seeds carry a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice aroma gene and a recessive homozygous rice thermosensitive sterility gene.

9. The breeding method according to claim 1, characterized in that: The target genes in step 5) are rice blast resistance gene, rice grain quality gene and rice aroma gene; The H0 generation flower culture strain carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice fragrance gene and a recessive homozygous rice thermosensitive sterility gene.

10. The breeding method according to claim 1, characterized in that: In step 6), the long-grain fragrant disease-resistant rice photothermosensitive genic male sterile line carries a positive homozygous rice blast resistance gene, a positive homozygous rice grain gene, a recessive homozygous rice fragrance gene and a recessive homozygous rice thermosensitive male sterile gene.

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