Method for cultivating heat-resistant broad-spectrum durable rice blast-resistant rice breeding material based on polygene polymerization

Abstract

The invention discloses a method for cultivating a heat-resistant broad-spectrum durable rice blast-resistant rice breeding material based on polygene polymerization. According to the method, a high-heat-resistance and high-dominance indica-japonica hybrid indica rice restorer line is selected as a recurrent parent, a breeding intermediate material for polymerizing complementary broad-spectrum rice blast resistance genes Pigm and Pi-jx is used as a donor parent, and polymerization of the heat-resistance and broad-spectrum lasting rice blast resistance genes Pigm and Pi-jx is realized through a method of combining molecular breeding with conventional breeding. Meanwhile, by selecting representative strains to carry out artificial inoculation identification of seedling blast and panicle blast, natural induction identification of a rice blast retransmission area and heat resistance identification, and by combining main agronomic trait evaluation in the whole growth period, a heat-resistant broad-spectrum durable rice blast-resistant breeding material is obtained. The breeding material bred by using the method disclosed by the invention has relatively strong heat resistance and broad-spectrum and lasting rice blast resistance, and the breeding process of heat-resistant and rice blast-resistant rice varieties is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of rice molecular breeding technology and relates to a method for cultivating heat-resistant, broad-spectrum, and long-lasting rice breeding materials with resistance to rice blast based on multi-gene aggregation. Background Technology

[0002] Rice is one of the main food crops for humankind. Global warming has exacerbated the impact of abiotic stresses (high temperature, drought, etc.) and biotic stresses (rice blast, bacterial blight, etc.) on crop yields. In recent years, with the frequent occurrence of abnormal weather, heat damage and rice blast outbreaks have caused large-scale reductions in rice production, becoming the most significant threat to food security. However, currently available breeding materials for rice production are either heat-resistant but have poor resistance to rice blast, or resistant to rice blast but not heat-resistant. There is a severe lack of breeding materials and resources that are both heat-resistant and resistant to rice blast. In addition, conventional breeding cycles are too long, making it difficult to synergistically improve heat resistance and rice blast resistance, which limits the development of target breeding materials that meet production needs. Therefore, it is urgent to utilize various breeding techniques to accelerate the development of heat-resistant, broad-spectrum rice blast-resistant breeding materials to curb the harm of high temperature damage and rice blast to rice production.

[0003] High temperatures and heat damage have become a core environmental stressor restricting high and stable rice yields. In recent years, with the intensification of global warming and the frequent occurrence of extreme heat events, the impact on rice production has expanded from a single reduction in yield to a multi-dimensional crisis including quality decline and regional adaptability challenges. Therefore, it is urgent to screen heat-resistant germplasm resources, especially those with excellent comprehensive traits that can be directly bred, and to utilize their heat-resistant characteristics to cultivate heat-resistant rice varieties to mitigate the impact of extreme high temperatures on rice production.

[0004] Rice blast, caused by *Magnaporthe oryzae*, is one of the most significant fungal diseases threatening rice production, causing a 10-30% annual reduction in rice yield globally (Skamnioti and Gurr 2009). Over the past 40 years, due to the narrow genetic base of parental lines and the diversity of highly pathogenic *Magnaporthe oryzae*, rice blast has become a major problem in rice production (Liu et al. 2010), posing a significant threat to rice production safety (Wu et al. 2016). Using marker-assisted selection (MAS) technology to aggregate different broad-spectrum, durable resistance genes against rice blast, and to breed new rice varieties with broad-spectrum, durable resistance to rice blast, is the most economical and effective measure for controlling rice blast and can achieve durable resistance in rice varieties.

[0005] The broad-spectrum rice blast resistance gene Pigm has been proven to have a broad resistance spectrum and strong resistance (Deng et al., 2006), and has good resistance effects against seedling blast and panicle blast (Wu et al., 2016). Its application value has been proven in basic research and production practice. However, in actual production applications, due to the many physiological races of rice blast fungus and the frequent differentiation and variation of pathogenicity, disease-resistant varieties bred using a single resistance gene often "lose" their resistance quickly and become susceptible varieties after a short period of application. After many years of production and application, the resistance of Pigm has also shown a gradual downward trend. Pi-jx is a newly discovered broad-spectrum rice blast resistance gene that exhibits good resistance against 155 pathogens collected from eight provinces including Jiangsu, Anhui, Hubei, Hunan, Henan, Zhejiang, Guangdong, and Shandong. Most importantly, it demonstrates excellent resistance against the highly pathogenic rice blast strain R5-1 (isolated from the Wuling Mountains, an area prone to severe rice blast, with a pathogenicity rate exceeding 95% across different genotypes, significantly higher than common rice blast strains isolated in large-scale production), which exhibits a broad-spectrum resistance gene for Pim. It has been confirmed that the resistance of Pi-jx has a complementary effect with that of Pim, compensating for the loss of resistance in Pim. Therefore, combining the rice blast resistance genes Pim and Pi-jx using marker-assisted selection will provide a new gene combination model for breeding broad-spectrum and durable blast-resistant rice varieties, and will also cultivate broader-spectrum and more durable resistant rice breeding materials to meet the needs of rice production.

[0006] Marker-assisted selection (MAS) uses molecular markers that are tightly linked to or co-segregate with a target gene to screen for that gene. Because it is unaffected by environmental conditions, it increases the reliability of selection and reduces the workload of breeding. In modern breeding, MAS has become a major approach for developing disease-resistant varieties, especially by utilizing already cloned disease-resistant genes, which increases the reliability of selection and reduces the workload and cost of breeding (Ishihara et al. 2014; Jiang et al. 2012; Narayanan et al. 2002). Using MAS to aggregate several or more disease-resistant genes has long been considered an effective way to develop broad-spectrum, durable disease-resistant varieties (Dai et al. 2007; Fukuoka et al. 2009; Jeung et al. 2007).

[0007] This invention utilizes molecular marker-assisted selection (MAS) technology to aggregate the broad-spectrum rice blast resistance genes Pigm and Pi-jx onto the large-scale production-use strong-advantage restorer line MR89 with indica-japonica lineage, thereby cultivating heat-resistant, broad-spectrum, and long-lasting rice breeding materials with resistance to rice blast. This is intended to address the high-temperature heat damage and rice blast epidemics faced in actual rice production, and effectively safeguard national food security. Summary of the Invention

[0008] The purpose of this invention is to address the problems existing in the prior art by providing a method for cultivating heat-resistant, broad-spectrum, and long-lasting rice breeding materials based on multi-gene aggregation.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] A method for breeding heat-resistant, broad-spectrum, and durable rice varieties with resistance to rice blast based on multi-gene aggregation, characterized by the following specific steps:

[0011] (1) Select breeding materials with excellent comprehensive agronomic traits and heat resistance and high temperature resistance verified on a large scale, but poor resistance to rice blast, as recipient parents, denoted as parent P1, as hybrid female parents and recurrent parents; select breeding intermediate materials that aggregate complementary rice blast resistance genes, or breeding intermediate materials that aggregate two rice blast resistance genes, denoted as parent P2, as rice blast resistance gene donor parents, as hybrid male parents; obtain F1 after hybridization of the two; wherein, the complementary rice blast resistance gene or the two rice blast resistance genes are broad-spectrum rice blast resistance gene Pigm and broad-spectrum rice blast resistance gene Pi-jx;

[0012] (2) Plant F1 generation seeds, backcross with the recurrent parents during the flowering period to obtain backcross generation 1 BC1F1 population;

[0013] (3) Plant the BC1F1 population with a planting scale of 100 plants. During the seedling stage, molecular detection was performed using the molecular marker InDel587, which is closely linked to the target resistance gene Pigm, and the KASP molecular marker SN285, which is closely linked to the target resistance gene Pi-jx. Heterozygous single plants containing two target resistance genes and with similar main agronomic traits and recurrent parents were selected. When the single plants were about to head, heat resistance was identified. The identification standard was based on DB 34 / T 3484-2019. Single plants with strong or extremely strong heat resistance were selected for stubble regeneration. They were backcrossed with the recurrent parents during the flowering period of the regenerated rice to obtain the BC2F1 population.

[0014] (4) Repeat step (3) above until backcrossing for 4 generations to obtain the BC4F1 population;

[0015] (5) Plant the BC4F1 population. During the seedling stage, the above linkage markers were used to perform molecular detection on the target resistance gene. During the heading stage, heat resistance was identified. Single plants with homozygous resistance genes, strong heat resistance, and main agronomic traits similar to the recurrent parents were selected for self-pollination and seed collection to obtain the BC4F2 line.

[0016] (6) Repeat step (5) above until the third generation of self-pollination is obtained to obtain the BC4F3 line;

[0017] (7) Plant the BC4F3 line. During the seedling stage, the above linkage markers are used to perform molecular detection and heat resistance identification on the target resistance gene. At the maturity stage, the seeds of the line with homozygous resistance gene, strong heat resistance, and similar main agronomic traits to the recurrent parent are harvested to obtain the BC4F4 line.

[0018] (8) Plant the BC4F4 population by line, and expand the planting scale to more than 500 plants. During the seedling stage, use the above linkage markers to detect the target resistance gene in individual plants in the population. During the booting stage, use the highly virulent monosporous strain R to identify the blast resistance of each line. During the heading stage, identify the heat resistance of each line. During the maturity stage, harvest the seeds of the line that is homozygous for the target gene, has strong resistance to rice blast, strong or very strong heat resistance, and agronomic traits similar to the recurrent parent to obtain BC4F5.

[0019] (9) The seeds of the harvested line BC4F5 were tested for seedling blast resistance, panicle blast resistance and heat resistance; at maturity, agronomic traits were evaluated, and lines resistant to seedling blast and panicle blast, with strong or very strong heat resistance and main agronomic traits similar to the recurrent parents were selected to obtain the heat-resistant and broad-spectrum rice blast-resistant breeding material BC4F6 with the aggregation of heat resistance and rice blast resistance genes.

[0020] (10) Identification of heat resistance and heat resistance of heat-resistant broad-spectrum and long-lasting rice blast resistance breeding material (BC4F6): Seven representative strains collected and isolated from different ecological regions and belonging to groups A, B, C, D, E, F and G (for Chinese rice blast identification varieties) were used to identify seedling blast and panicle blast resistance by single-line inoculation. At the same time, rice blast disease nurseries in multiple locations were identified to comprehensively evaluate the overall resistance of the breeding material. Heat resistance identification was carried out in accordance with reference standard DB 34 / T 3484-2019.

[0021] (11) Verification of heat resistance and heat tolerance of the heat-resistant broad-spectrum and long-lasting rice blast resistance breeding material (BC4F7): 150 single-celled strains collected and isolated from southern rice-growing areas such as Sichuan, Chongqing, Hubei, Jiangxi, Anhui, Hunan, Guangdong, Hainan, Fujian, and Jiangsu were used for single-line inoculation identification of the heat-resistant broad-spectrum and long-lasting rice blast resistance breeding material (BC4F7) and recurrent parents in the seedling stage and during the booting stage. At the same time, natural induction identification was carried out in rice blast-prone areas such as Enshi in Hubei, Jinggangshan in Jiangxi, Shanghang in Fujian, and Jinzhai in Anhui. In addition, 30 single-celled strains (10 from each province) were selected from the rice blast fungus bank collected and preserved for many consecutive years and were used for single-line inoculation identification by injection during the booting stage. During the same period, the heat tolerance of the breeding material was verified by artificial climate chamber identification and high-temperature verification in production practice. The heat tolerance identification was carried out in accordance with the reference standard DB 34 / T3484-2019.

[0022] The recipient parent, i.e. the recurrent parent, is MR89, which has strong heat resistance and hybrid vigor between indica and japonica rice; the donor parent, YD6-Pigm / Pi-jx, has broad-spectrum and long-lasting resistance to rice blast.

[0023] The broad-spectrum, long-lasting resistance to rice blast gene Pi-jx has a complementary effect with Pigm resistance.

[0024] The preferred molecular marker closely linked to the target gene Pigm is InDel587, and the preferred molecular marker for identifying the target gene Pi-jx is the KASP molecular marker SN285.

[0025] The primers for the molecular marker InDel587 are based on the national invention patent (InDel587, a molecular marker for detecting the gene Pigm(t) of rice blast resistance in Gumei 4, invention patent number: ZL 2013 1 0428162.0).

[0026] The molecular marker SN285 primers are referenced from the national invention patent (a method for breeding broad-spectrum and durable resistant rice breeding materials by polymerizing complementary anti-blast rice genes, invention patent number: ZL2021 1 0071094.1).

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1) The recipient parent used in this invention is the highly heat-resistant, strong-hypergenic indica-japonica hybrid rice restorer line MR89, which has the following advantages: First, it has extremely strong heat resistance; hybrid indica rice bred with it as the male parent exhibits exceptional heat resistance and can fully cope with the damage caused by extreme high temperatures to large-scale rice production. Second, it possesses japonica rice lineage, allowing for the full realization of indica-japonica hybrid vigor and exhibiting ultra-high yield potential. Using it as the male parent to breed hybrid indica rice plays a crucial role in ensuring national food security. However, MR89 has extremely poor resistance to rice blast; only by improving its rice blast resistance can its strong heat resistance and indica-japonica hybrid vigor potential be fully realized.

[0029] 2) The donor parents used in this invention are breeding intermediates of the polymerized complementary rice blast resistance genes Pigm and Pi-jx, which have the following advantages: strong resistance to rice blast, broad resistance spectrum, and long-lasting resistance. The broad-spectrum rice blast resistance gene Pigm is generally recognized as having broad-spectrum resistance, but after large-scale application, new pathogenic strains have emerged. Pi-jx, on the other hand, is a newly discovered broad-spectrum rice blast resistance gene that shows resistance to new pathogenic strains resistant to Pigm. Theoretically, it should have a complementary effect with Pigm, compensating for the loss of Pigm resistance. The polymerized complementary rice blast resistance gene "Pigm+Pi-jx" can better address the complex physiological races of rice blast fungus, frequent pathogenicity differentiation and variation in production, and avoids the defect of disease-resistant varieties bred using a single resistance gene quickly "losing" resistance after a short period of application.

[0030] 3) The recipient parent and donor parent used in this invention have excellent overall agronomic traits, in addition to the strong heat resistance and strong indica-japonica advantages of the recipient parent and the broad-spectrum and long-lasting resistance to rice blast of the donor parent. There are no unfavorable linkage burdens, which avoids the drawbacks of using resistance resources (poor overall agronomic traits, linkage burdens, long time required to break linkage burdens, and difficulty in direct production and application). In the process of improving by using molecular markers, the offspring traits are stable and fast, which can accelerate the breeding process, shorten the cultivation time, cope with the recent continuous high temperature extreme climate and rice blast damage, and meet the urgent needs of large-scale rice production.

[0031] 4) This invention conducts field tests on the resistance and heat tolerance of the heat-resistant, broad-spectrum rice breeding materials bred for rice blast in seedling blast, panicle blast, and severely diseased nurseries, and rates their comprehensive resistance. The advantage is that the heat-resistant rice blast-resistant materials or lines bred can withstand the test of large-scale rice production practice. Attached Figure Description

[0032] Figure 1 A schematic diagram of the technical route for breeding heat-resistant, broad-spectrum rice varieties resistant to rice blast based on multi-gene aggregation. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to specific embodiments.

[0034] Example:

[0035] 1. Recipient parent and rice blast resistance gene donor parent

[0036] The recipient parent is the highly heat-resistant, strong-advantage indica-japonica hybrid rice restorer line MR89 (an indica rice restorer line bred by the Xuancheng City Planting Industry Bureau, operated by Anhui Yuanliang Rice Industry Co., Ltd., available for purchase from Anhui Yuanliang Rice Industry Co., Ltd., or searchable in the National Rice Data Center <National Rice Data Center == RiceData == Chinese Rice Varieties and Their Pedigrees and Fingerprint Database>); the donor parent is the breeding intermediate material YD6-Pigm / Pi-jx, which is a composite complementary rice blast resistance gene Pigm and Pi-jx (National Invention Patent: A method for cultivating broad-spectrum, durable resistant rice breeding materials using composite complementary rice blast resistance genes, Invention Patent No.: ZL2021 1 0071094.1).

[0037] 2. The process of cultivating heat-resistant, broad-spectrum, and long-lasting rice breeding materials based on multi-gene aggregation:

[0038] (1) Using the heat-resistant indica-japonica hybrid strong dominant indica rice restorer line MR89 as the recipient, and YD6-Pigm / Pi-jx, a breeding intermediate material carrying complementary broad-spectrum resistance to rice blast genes Pigm and Pi-jx, as the donor, hybridization was carried out to obtain F1.

[0039] (2) Plant F1 generation seeds and backcross with the recurrent parent MR89 during the flowering period to obtain the backcross generation BC1F1 population.

[0040] (3) Plant the BC1F1 population with a planting scale of 100 plants (the same below). During the seedling stage, molecular detection was performed using the molecular marker InDel587, which is closely linked to the broad-spectrum rice blast resistance gene Pigm, and the KASP marker SN285, which is closely linked to the resistance gene Pi-jx. Heterozygous single plants containing the two target resistance genes and with similar main agronomic traits and recurrent parents were selected. When the single plants were about to head, heat tolerance was identified, and the identification standard referred to DB 34 / T 3484-2019. Single plants with strong or extremely strong heat tolerance were selected for stubble regeneration and backcrossed with the recurrent parents at the flowering stage of the ratooning rice to obtain the BC2F1 population. The upstream primer for detecting molecular marker InDel587 is 5'-AACTTGCTGGGAGAAGGATTG-3' (SEQ ID NO.1), and the downstream primer is 5'-GAGTTCGTACTTTTCAGGCTT-3' (SEQ ID NO.2). For detecting molecular marker SN285, the forward primer Pi-jx-FG sequence for SN285 is 5'-GAAGGTGACCAAGTTCATGCTTCCATAGCAGGGACCCTACAG-3' (SEQ ID NO.3), the forward primer Pi-jx-FC sequence is 5'-GAAGGTCGGAGTCAACGGATTTTCCATAGCAGGGACCCTATAA-3' (SEQ ID NO.4), and the reverse universal primer Pi-jx-R sequence is 5'-GCAGAGCACACCATACCCAC-3' (SEQ ID NO.5).

[0041] (4) Repeat step (3) above until backcrossing for 4 generations to obtain the BC4F1 population.

[0042] (5) Plant the BC4F1 population. During the seedling stage, molecular detection of the target resistance gene was carried out using the above linkage markers. During the heading stage, heat resistance was identified. Single plants with homozygous resistance genes, strong heat resistance, and main agronomic traits similar to the recurrent parents were selected for self-pollination and seed collection to obtain the BC4F2 line.

[0043] (6) Repeat step (5) above until the third generation of self-pollination is obtained to obtain the BC4F3 line.

[0044] (7) Plant the BC4F3 line. During the seedling stage, the above linkage markers are used to perform molecular detection and heat resistance identification on the target resistance gene. At the maturity stage, the seeds of the line with homozygous resistance gene, strong heat resistance, and similar main agronomic traits to the recurrent parent are harvested to obtain the BC4F4 line.

[0045] (8) Plant the BC4F4 line separately, and expand the planting scale to more than 500 plants. During the seedling stage, use the above linkage markers to detect the target resistance gene in each plant in the population. During the booting stage, use the highly virulent monosporous strain R to identify the blast resistance of each line. During the heading stage, identify the heat resistance of each line. At the maturity stage, harvest the lines that are homozygous for the target gene, have strong resistance to rice blast, and have strong or extremely strong heat resistance to obtain the BC4F5 line.

[0046] (9) Seeds of the harvested line BC4F5 were inoculated indoors during the seedling stage to identify seedling blast resistance; in the field, mixed inoculation was performed by injection during the booting stage to identify panicle blast resistance; heat tolerance was identified during the heading stage; and agronomic traits were evaluated at the maturity stage. Lines resistant to seedling blast and panicle blast, with strong or very strong heat tolerance, and whose main agronomic traits are similar to the recurrent parents were selected to obtain a heat-resistant, broad-spectrum blast-resistant breeding material (BC4F6) that integrates heat tolerance and blast resistance genes.

[0047] (10) Resistance identification of heat-resistant, broad-spectrum, and long-lasting rice blast resistance breeding material (BC4F6). Seven representative strains collected and isolated from different ecological regions and belonging to groups A, B, C, D, E, F, and G (for identifying rice blast varieties in China) were used to identify the seedling blast and panicle blast resistance of the bred broad-spectrum and long-lasting rice blast resistance breeding material (BC4F6). For indoor seedling inoculation and identification methods, refer to Yu Miaomiao (2013) (Yu Miaomiao et al., Differences in resistance spectrum between broad-spectrum rice blast resistance genes Pigm and Pi2 and their interaction with Pi1. Acta Agronomica Sinica, 2013, 39(11):1927-1934); For mixed inoculation at the booting stage, refer to Luo Chuping (2009) (Luo Chuping et al., Rice blast inoculation technology and resistance identification of varieties in Jiangsu Province regional trials in 2009. Jiangsu Agricultural Sciences, 2009, 6: 178-179); At the same time, identification was carried out in rice blast disease nurseries in multiple locations (Jinggangshan, Jiangxi; Enshi, Hubei; Jinzhai, Anhui, etc.) to comprehensively evaluate the overall resistance of the breeding materials. Heat resistance identification was still carried out in accordance with the reference standard DB 34 / T 3484-2019. At maturity, the main agronomic traits of each line were investigated, including plant height, growth period and yield composition traits. Based on the combined results of seedling blast, panicle blast resistance, disease nursery resistance and heat tolerance identification, the breeding material MR89-Pigm / Pi-jx (BC4F7) was finally obtained, which has aggregated heat tolerance and aggregated broad-spectrum and durable resistance genes Pigm and Pi-jx to rice blast.

[0048] (11) Verification of the resistance and heat resistance of the heat-resistant, broad-spectrum, and long-lasting rice blast resistance breeding material MR89-Pigm / Pi-jx (BC4F7). Using 150 single-celled strains collected and isolated from rice-growing areas in southern China, including Sichuan, Chongqing, Hubei, Jiangxi, Anhui, Hunan, Guangdong, Hainan, Fujian, and Jiangsu, single-line inoculation identification was conducted on the heat-resistant, broad-spectrum, and durable rice blast-resistant breeding material MR89-Pigm / Pi-jx and the recurrent parent MR89 using indoor inoculation methods during the seedling stage and the booting stage. The results showed that the seedling blast resistance frequency was as high as 98±1.26%, and the panicle blast resistance frequency was 97±2.15%, while the recurrent parent's were only 11.55±1.96% and 16.75±3.51%, respectively. Natural induction identification was carried out on MR89-Pigm / Pi-jx and MR89 in severely blast-affected areas such as Enshi in Hubei, Jinggangshan in Jiangxi, Shanghang in Fujian, and Jinzhai in Anhui. The resistance evaluation standard adopted was the International Rice Research Institute (IRRI). (2002. Standard Evaluation System for Rice (SES). 2002, (4th ed., pp. 15-16). Los Banos.) Philippines: Following the 0-9 grading standard established by the International Rice Research Institute (IRRI), the results showed that MR89-Pigm / Pi-jx had a blast disease grade below level 3 in all four disease nurseries, reaching a resistance level, while MR89 had a grade above level 8.6 in all four nurseries, showing high susceptibility. Furthermore, from 2022 to 2024, 30 single-celled strains (10 from each of Hubei, Anhui, and Jiangsu provinces) were selected annually from the collected and preserved rice blast pathogen bank. During the booting stage, single-line inoculation identification of MR89-Pigm / Pi-jx and MR89 was performed using the injection method. The results showed that the blast resistance frequency of MR89-Pigm / Pi-jx remained stable above 90%, while that of MR89 was below 10%. Concurrently, heat resistance assessment in artificial climate chambers and high-temperature verification in production practice showed that the heat resistance levels of MR89-Pigm / Pi-jx and MR89 remained stable between 1.9 and 2.0, respectively. Particularly noteworthy were the exceptionally high-temperature years of 2022 and 2024, where the field seed setting rate of MR89-Pigm / Pi-jx exceeded 85%. These comprehensive results demonstrate that the heat-resistant and blast-resistant breeding material MR89-Pigm / Pi-jx possesses broad-spectrum and durable blast resistance and strong heat resistance, fully proving the effectiveness of this invention.

Claims

1. A method for breeding heat-resistant, broad-spectrum, and durable rice varieties with resistance to rice blast based on multi-gene aggregation, characterized by the following specific steps: (1) Select breeding materials with excellent comprehensive agronomic traits and proven to be heat-resistant and high-temperature resistant on a large scale, but with poor resistance to rice blast, as recipient parents, denoted as parent P1, serving as the female parent and recurrent parent; select intermediate breeding materials that aggregate complementary rice blast resistance genes, or intermediate breeding materials that aggregate two rice blast resistance genes, denoted as parent P2, serving as the donor parent of the rice blast resistance gene, serving as the male parent; after hybridization, obtain F1; among which, The complementary rice blast resistance gene or the two rice blast resistance genes mentioned are broad-spectrum rice blast resistance genes. Pigm and broad-spectrum resistance to rice blast genes Pi-jx ; (2) Plant F1 generation seeds, backcross with the recurrent parents during the flowering period to obtain backcross generation 1 BC1F1 population; (3) Plant the BC1F1 population at a scale of 100 plants, and utilize the target resistance gene during the seedling stage. Pigm Tightly linked molecular marker InDel587 and the target resistance gene Pi-jx Molecular detection was performed using the tightly linked KASP molecular marker SN285. Heterozygous single plants containing two target resistance genes and with similar main agronomic traits and recurrent parents were selected. When the single plants were about to head, heat tolerance was assessed according to DB 34 / T 3484-2019. Single plants with strong or extremely strong heat tolerance were selected for stubble regeneration and backcrossed with the recurrent parents at the flowering stage of the ratooning rice to obtain the BC2F1 population. (4) Repeat step (3) above until backcrossing for 4 generations to obtain the BC4F1 population; (5) Plant the BC4F1 population. During the seedling stage, the above linkage markers were used to perform molecular detection on the target resistance gene. During the heading stage, heat resistance was identified. Single plants with homozygous resistance genes, strong heat resistance, and main agronomic traits similar to the recurrent parents were selected for self-pollination and seed collection to obtain the BC4F2 line. (6) Repeat step (5) above until the third generation of self-pollination is obtained to obtain the BC4F3 line; (7) Plant the BC4F3 line. During the seedling stage, the above linkage markers are used to perform molecular detection and heat resistance identification on the target resistance gene. At the maturity stage, seeds of the line with homozygous resistance gene, strong heat resistance, and main agronomic traits similar to the recurrent parent are harvested. (8) Plant the BC4F4 population by line, and expand the planting scale to more than 500 plants. During the seedling stage, use the above linkage markers to detect the target resistance gene in individual plants in the population. During the booting stage, use the highly virulent monosporous strain R to identify the blast resistance of each line. During the heading stage, identify the heat resistance of each line. During the maturity stage, harvest the seeds of the line that is homozygous for the target gene, has strong resistance to rice blast, strong or very strong heat resistance, and agronomic traits similar to the recurrent parent to obtain BC4F5. (9) The seeds of the harvested line BC4F5 were tested for seedling blast resistance, panicle blast resistance and heat resistance; at maturity, agronomic traits were evaluated, and lines resistant to seedling blast and panicle blast, with strong or very strong heat resistance and main agronomic traits similar to the recurrent parents were selected to obtain the heat-resistant and broad-spectrum rice blast-resistant breeding material BC4F6 with the aggregation of heat resistance and rice blast resistance genes. (10) Identification of heat resistance and heat tolerance of BC4F6, a heat-resistant, broad-spectrum, and long-lasting rice blast resistance breeding material: Seven representative strains collected and isolated from different ecological regions and belonging to groups A, B, C, D, E, F, and G (for identifying rice blast varieties in China) were used to identify the seedling blast and panicle blast resistance of BC4F6. At the same time, rice blast disease nurseries in multiple locations were identified to comprehensively evaluate the overall resistance of the breeding material. The heat tolerance was identified in accordance with the reference standard DB 34 / T3484-2019.

2. (11) Verification of the heat resistance and heat tolerance of the heat-resistant broad-spectrum and long-lasting rice blast resistance breeding material BC4F7: 150 single-cell strains collected and isolated from southern rice-growing areas such as Sichuan, Chongqing, Hubei, Jiangxi, Anhui, Hunan, Guangdong, Hainan, Fujian, and Jiangsu were used to identify the heat-resistant broad-spectrum and long-lasting rice blast resistance breeding material BC4F7 and its recurrent parents by injection during the seedling stage and the booting stage. At the same time, natural induction identification was carried out in rice blast-prone areas such as Enshi in Hubei, Jinggangshan in Jiangxi, Shanghang in Fujian, and Jinzhai in Anhui. In addition, 30 single-cell strains from Hubei, Anhui, and Jiangsu provinces were selected from the rice blast fungus bank collected and preserved for many consecutive years and identified by injection during the booting stage. At the same time, the heat tolerance of the breeding material was verified by artificial climate chamber identification and high temperature verification in production practice. The heat tolerance identification was carried out with reference to standard DB 34 / T 3484-2019.

3. The method according to claim 1, characterized in that: The breeding material with excellent comprehensive agronomic traits and proven heat resistance and high-temperature tolerance in large-scale production, but poor resistance to rice blast, is the strong-yielding indica-japonica hybrid rice restorer line MR89. The intermediate breeding material for the aggregation and complementation of rice blast resistance genes is YD6- Pigm / Pi-jx .

4. The method for cultivating heat-resistant, broad-spectrum, and long-lasting rice breeding materials based on multi-gene aggregation according to claim 1, characterized in that: In steps (9) and (11), seedling resistance was assessed by indoor inoculation during the seedling stage; panicle resistance was assessed by mixed inoculation via injection during field planting and the booting stage; rice blast resistance was assessed by comprehensive evaluation of multiple disease nurseries throughout the entire growth period; heat tolerance was assessed during the heading stage; and the heat-resistant, broad-spectrum, and long-lasting rice blast-resistant breeding material MR89- was comprehensively evaluated. Pigm / Pi-jx It exhibits heat resistance and resistance to rice blast.

Images