CN115491430B

CN115491430B - Rice blast multiple disease-resistant gene combination and application thereof

Info

Publication number: CN115491430B
Application number: CN202211360005.6A
Authority: CN
Inventors: 齐中强; 刘永锋; 杜艳
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2023-09-08
Anticipated expiration: 2042-11-02
Also published as: CN116949205A; CN116949206A; CN115491430A; CN116949206B; CN116987813A; CN116949205B; CN116987813B

Abstract

The application belongs to the technical field of rice genetic engineering, and particularly relates to a rice blast multiple disease-resistant gene combination and application thereof. The application carries out comprehensive evaluation on the leaf blast and spike blast resistance of 119 main rice cultivars in Jiangsu province for 3 years continuously, and determines the resistance level of the main rice cultivars to the rice blast; and detecting the distribution frequency of 14 disease resistance genes by using SSR molecular markers. The major resistance genes and gene combination patterns suitable for Jiangsu rice varieties are determined by utilizing gene resistance contribution rate and multiple stepwise regression analysis, specifically i) Pita+Pikm+Piz-t, ii) Pita+Pikm+Pi2, iii) Pita+Pi5+Piz-t, iv) Pikm+Piz-t+Pi2, v) Pita+Pikm+Pi5+Piz-t, vi) Pita+Pikm+Pi2+Piz-t. The resistance gene combination can effectively improve the resistance of rice varieties.

Description

Rice blast multiple disease-resistant gene combination and application thereof

Technical Field

The application belongs to the technical field of rice genetic engineering, and particularly relates to a rice blast multiple disease-resistant gene combination and application thereof.

Background

Rice (Oryza sativa) is one of the most important staple foods worldwide, accounting for 23% of the heat consumed by 50% of the population worldwide. The safe production of rice is severely threatened by rice blast, which is caused by ascomycetes (Magnaporthe oryzae) and causes 10-30% of world grain loss every year. Because of the transition zone of the north and south climates, the Jiangsu paddy rice producing area is a rice blast frequent area. The prevention and control area per year is about 200 ten thousand hectares, and the safety production of rice is seriously threatened. The selection of disease-resistant varieties is the most economical and effective method for controlling the disease. However, due to the complexity of the physiological race composition of Pyricularia oryzae and the variability of non-toxic genes, disease resistant varieties will have reduced or lost resistance after 3-5 years of field cultivation. Therefore, the identification of the resistance of the main cultivar to leaf blast and spike blast and the analysis of the distribution of the resistance genes are key to improving the rice resistance.

In the rice/Pyricularia oryzae interaction system, the disease-resistant gene in the rice and the Pyricularia oryzae nontoxic gene follow the gene-to-gene hypothesis, and the rice variety containing the disease-resistant gene can effectively inhibit the infection of Pyricularia oryzae containing the corresponding nontoxic gene. Up to now, more than 100 rice blast resistance genes have been identified, of which 25 major disease resistance genes and 2 partial resistance genes (Pi 21 and Pb 1) have been cloned or identified. All disease resistance genes cloned (except Pid2 and Pi 21) belong to the nucleotide binding site and leucine rich repeat (NBS-LRR) classes. Pita is located on chromosome 12, encodes a plasma membrane receptor protein, recognizes with the avirulence gene Avr-Pita of Magnaporthe grisea, and the expressed product interacts to induce a resistance response. At the Pik locus 7 alleles Pik, pikp, pikh, pikm, piks, pike and Pi1 were identified, which required two adjacent NBS-LRR genes to function fully. Another disease resistance gene locus is Piz locus, which includes 5 disease resistance genes Pi9, pi2, piz-t, pigm and Pi 50. Wherein Pigm encodes two proteins, pigmR and PigmS, together regulate broad-spectrum durable resistance and yield.

The traditional breeding method is to introduce a resistance gene into a target line through hybridization, agronomic character selection and resistance identification to form a resistance variety, and the method is effective, but has large workload and long time consumption. Molecular Marker Assisted Selection (MAS) is a selection assistance method that applies molecular markers to rice breeding. Functional markers (Functional markers, FMs) refer to marker loci representing specific resistance genes, and rice traits can be screened by screening for molecular markers. Some FMs for disease resistance genes have been developed, such as Pib, pigm, pita and pitm, which would provide a convenient method for identifying target genes. In addition, some PCR-based tight Link Markers (LMs) are associated with multiple disease resistance genes, including Pi2, pi5, and Pi 9. The LMs provides a high-efficiency and rapid method for screening target genes in gene introgression and gene polymerization.

The rice blast is classified into leaf blast and spike blast according to the pathogenesis, and the spike blast is more destructive in terms of yield loss. There is increasing evidence that there is a different regulatory mechanism between leaf blast resistance and ear blast resistance. It is well known that gene polymerization can increase the resistance spectrum and level of resistance of rice varieties. Liu et al reported that Pita2, pi5, pi9 and Piz-t exhibited higher frequencies and resistances in 56 major varieties of rice in Yunnan. In addition, the "Pi9+Pi54", "Pid3+Pigm", "Pi5+Pid3+Pigm", "Pi5+Pi54+Pid3+Pigm" and the "Pi5+Pib", "pik+Pita", "pik+Pb1", "Piz-t+Pia" and "Piz-t+Pita" combination patterns in the indica type combination are critical to the resistance of rice varieties. However, these studies are all based on leaf blast resistance, and the accuracy in breeding applications is not high. The breeding and utilizing of disease-resistant varieties are the most economical and effective measures for preventing and controlling rice blast and epidemic, so that a more accurate disease-resistant gene combination is needed to improve the breeding efficiency of the disease-resistant varieties.

Disclosure of Invention

In order to solve the technical problems, the application comprehensively evaluates the resistance of 119 main rice cultivars in Jiangsu province to leaf blast and spike blast for 3 years continuously, and determines the resistance level of the main rice cultivars to rice blast; the distribution frequency of 14 disease resistance genes is detected by utilizing SSR molecular markers. The main disease-resistant genes and the gene combination modes suitable for Jiangsu rice varieties are determined by utilizing the contribution rate of the gene resistance and multiple stepwise regression analysis. The comprehensive resistance index of leaf blast and spike blast is used for analyzing the disease resistance gene distribution and the disease resistance level of the main cultivated rice variety in the main production area of Jiangsu province, and has important significance for accurately utilizing the disease resistance genes/combinations.

In a first aspect, the present application provides a combination of rice blast multiple disease-resistant genes selected from any one of the following combinations: i) Pita+Pikm+Piz-t, ii) Pita+Pikm+Pi2, iii) Pita+Pi5+Piz-t, iv) Pikm+Piz-t+Pi2, v) Pita+Pikm+Pi5+Piz-t, vi) Pita+Pikm+Pi2+Piz-t.

In a second aspect, the application also provides the application of the rice blast multiple disease resistance gene combination in any one of the following,

a) Selecting or screening rice varieties with broad-spectrum durable rice blast resistance;

b) Comparing the resistance of the rice blast to be detected;

c) Selecting or screening rice varieties with relatively strong rice blast resistance.

In a third aspect, the application also provides the application of the reagent for detecting the rice blast multiple disease resistance gene combination in preparing a product with any one of the following functions,

e) Selecting or screening rice varieties with broad-spectrum durable rice blast resistance;

f) Comparing the resistance of the rice blast to be detected;

g) Selecting or screening rice varieties with relatively strong rice blast resistance.

In certain embodiments, the product is a detection kit.

In a fourth aspect, the application also provides any one of the following methods,

a) Selecting or screening rice varieties with broad-spectrum durable rice blast resistance, comprising the following steps:

(A1) Extracting genome DNA of rice to be detected,

(A2) Performing PCR amplification on genome DNA of rice to be detected, wherein if the genome DNA contains at least one of the rice blast multiple disease resistance gene combinations, the rice to be detected is a rice variety with broad-spectrum durable rice blast resistance;

b) Comparing the resistance of the rice blast to be detected, comprising the following steps:

(B1) Extracting genome DNA of rice to be detected,

(B2) Carrying out PCR amplification on genome DNA of rice to be detected, wherein the rice blast resistance of a rice variety containing at least one of the rice blast multiple resistance gene combinations in the genome DNA is stronger than that of a rice variety containing no rice blast multiple resistance gene combination in the genome DNA;

c) Selecting or screening rice varieties with relatively strong rice blast resistance, comprising the following steps:

(C1) Extracting genome DNA of rice to be detected,

(C2) Performing PCR amplification on genome DNA of rice to be detected, wherein the genome DNA contains at least one of the rice blast multiple disease resistance gene combinations, and the rice to be detected is a rice variety with relatively strong rice blast resistance.

In certain embodiments, the rice variety is Jiangsu province rice variety.

Compared with the prior art, the application has the beneficial effects that:

1. the application discovers that the resistance level of the rice variety has a weak positive correlation with the number of resistance genes, so that the more the number of disease resistance genes is, the higher the resistance level of the rice variety is, and the best strategy is to aggregate effective resistance genes. Combining multiple stepwise regression and resistance contribution rate data, 6 gene combinations were screened for resistance contribution rates of 100% each, i) pita+pitm+piz-t, ii) pita+pitm+pi2, iii) pita+pi5+piz-t, iv) pitm+piz-t+pi2, v) pita+pikm+pi5+piz-t, vi) pita+pitm+pi2+piz-t, respectively.

2. According to the application, 119 representative main cultivated varieties are used as research materials, and the comprehensive indexes such as leaf blast, neck blast, ear loss index and the like are subjected to resistance evaluation through natural induction in the field, and the resistance evaluation is continuously carried out for 3 years, so that the combined result of the screened disease-resistant genes is more accurate and strict, and the true resistance of the varieties in the field can be reflected; meanwhile, by utilizing analysis methods such as a multiple stepwise regression model and resistance contribution rate, the disease resistance gene and gene combination with the maximum resistance correlation are more accurately determined, and the analysis result is more accurate.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.

FIG. 1 shows gel electrophoresis patterns of 14 disease resistance gene markers of partial varieties in example 1;

FIG. 2 evaluation of resistance and disease resistance gene analysis of rice varieties;

FIG. 3 is a cluster analysis chart of 119 major varieties of rice in example 4;

FIG. 4 analysis of the distribution frequency and contribution rate of resistance of 14 disease resistance genes in example 5.

Detailed Description

Embodiments of the technical scheme of the present application will be described in detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present application, and thus are merely examples, which should not be construed as limiting the scope of the present application. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs.

Materials and methods

The application collects 119 main varieties of rice (94 japonica rice and 25 indica rice) from all planting areas (Subei, suzhong and Sunan) in Jiangsu province.

1. Analysis of rice infection

The application adopts a gold altar (31 DEG 40 '20' N,119 DEG 21 '34' E) nursery to naturally induce leaf blast and spike blast. Each variety is planted with 25 (5×5) holes, 1 square meter in total, each row (5 holes), and the susceptible variety Co39 is planted between varieties and at the periphery of the variety to promote the full onset of the variety. The leaf blast disease condition was investigated 40 days after sowing or when disease symptoms were present in susceptible varieties (85%). After 140 days or 150 days (yellow ripe period) of sowing, the occurrence of the spike plague of each variety was investigated.

2. Disease assessment

The severity of leaf blast is evaluated by adopting standard grade 0-9 and is divided into 10 grades, wherein the grade 0 is defined as no disease symptoms; grade 1, brown spots with the diameter less than or equal to 1mm; grade 2, large brown spots, diameter 1-2 mm; grade 3, circular to elliptical grey lesions, diameter 1-2cm; stage 4, spindle spots, 1-2cm long, limited between two veins, infection area not exceeding 2% of leaf area; stage 5, spindle spots, wherein the infection area accounts for 2% -10% of the leaf area; stage 6, spindle spots, wherein the infection area accounts for 11% -25% of the leaf area; stage 7, spindle spots, the infection area accounts for 26% -50% of the leaf area; stage 8, spindle spots, the infection area occupies 51% -75% of the leaf area; stage 9 spindle spots, the infection area is more than 75% of the leaf area.

The spike blast disease onset reaction is classified into 0-9 grades and 6 grades, and is defined as grade 0, no disease symptoms; grade 1, single branch is ill, and loss per ear is less than 5%; grade 3, about 1/3 of branches are ill, and the loss of each ear is 6% -20%; grade 5, the rice ears or the cobs are ill, and the loss of each ear is 21% -50%; grade 7, namely 51% -70% of loss of each spike of rice spike disease; grade 9, loss of 71% -100% per spike.

The incidence rate of the spike plague is graded as 0 grade, and no symptoms exist; grade 1, the incidence rate of spike blast is less than 5.0 percent; grade 3, the incidence rate of spike blast is 5.1-10.0%; grade 5, the incidence rate of spike blast is 10.1 to 25.0 percent; grade 7, the incidence rate of the spike blast is 25.1-50.0%; grade 9, the incidence rate of the spike blast is 50.1-100.0%. The incidence of spike blast= (number of diseased spikes/number of spikes investigated) ×100%.

The spike plague grading loss index standard is grade 0, and no disease symptoms exist; grade 1, loss of spike <5.0%;3 grade, the ear part falls off 5.1% -15.0%; grade 5, 15.1% -30.0%;7 grade, the ear part falls off 30.1% -50.0%; grade 9, 50.1% -100%.

The rice blast grading comprehensive index standard is 0 grade <0.1;1 grade, 0.1-2.0;3 grade 2.1-4.0;5 grade, 4.1-6.0; grade 7, 6.1-7.5; grade 9, 7.6-9.0. Comprehensive index = leaf blast level x 25% + incidence of ear blast x 25% + loss index of ear blast x 50%.

3. Molecular screening of rice blast disease-resistant genes

In molecular screening, 14 major rice blast resistant genes Pit, pish, pib, pi1, pia, pi54, pita, pi9, pi2, pikm, pigm, pi, pb1 and Piz-t were genotyped. 14 molecular markers were selected from the published primer sequences for molecular screening. The detailed information of the primer pairs is shown in Table 1.

TABLE 1 molecular marker detection primer sequences

The PCR amplification was 20. Mu.l, 10. Mu.l Green Taq Mix (Vazyme), 1. Mu.l (20 ng) template DNA, 1. Mu.l each primer, ddH ₂ O7. Mu.l. The PCR procedure was set as follows: initial denaturation at 95℃for 5min;35 cycles, 95℃30s,58℃30s,72℃30s; then put into 72℃for 10 minutes. The PCR products were separated by electrophoresis on a 3% agarose gel and run at 80 volts for 60 minutes. The gel image was photographed. PCR amplified fragments were scored for presence (1) and absence (0).

4. Data analysis

For diversity analysis, binary matrices of 14 resistance genes were used as binary data of presence (1) and absence (0) for estimating genetic distances and similarity coefficients. The data matrix was further analyzed using NTSYS-pc v.2.1. Jaccard similarity coefficients were calculated using the SIMQUAL program. The resulting similarity matrix is used to construct a dendrogram based on the non-weighted pair group method of arithmetic average (UPGMA).

Multiplex stepwise regression analysis was performed using SPSS 20 statistical software for the relationship of the resistance level to the disease gene. The average value of the comprehensive index of each variety over 3 years was used as the resistance level.

EXAMPLE 1 genotyping 14 disease resistance genes of Rice major cultivar

And (5) carrying out resistance gene identification by utilizing SSR molecular markers. The gel electrophoresis pattern of the 14 disease resistance gene markers of part of varieties is shown in figure 1. Pit, pish, pib, pita, pb1 and Pi97 disease resistance genes can be easily identified according to molecular markers. Since molecular markers of 7 genes such as Pi54, pi2, pi1, pi5, pikm, pia, piz-t/Pi2 and Pigm can amplify 2-3 bands, the identification of these genes is mainly based on specific electrophoresis bands (FIG. 1).

EXAMPLE 2 Rice blast phenotype analysis

According to the screening composite index scores for leaf and ear blast (Table 2), indica and japonica rice had 23 (92.00%) and 28 (29.79%) varieties with resistance and moderate resistance (grade 1 and grade 3), respectively, and 50 (53.19%) varieties with moderate susceptibility (FIG. 2A). The above results indicate that indica rice has better resistance to rice blast than japonica rice, and that the risk of degeneration of japonica rice resistance is greater. The 4 varieties of japonica rice (suxiang japonica 100, jinxiangyu No. 1, jia 58 and Shanghai LPR 18) and the 4 varieties of indica rice (long two you 1307, and two you 332, hui two you 882 and two you 688) were better resistant (Table 2). In addition, all 119 rice varieties were divided into thresh (74), thresh (21) and thresh (24) 3 rice planting areas (table 3), 50.00% of the rice varieties in the thresh area were resistant and medium resistant, which was higher than 44.60% in the thresh area and 28.57% in the thresh area (table 3), indicating that there was a significant difference in the resistance of the rice varieties in different producing areas to rice blast.

TABLE 2 identification and analysis of resistance of 119 main cultivars 2019-2021 of rice

TABLE 3 evaluation of regional resistance of 119 varieties

EXAMPLE 3 positive correlation of resistance level with resistance Gene

The number of the detected disease-resistant genes in the main rice varieties is normally distributed, and the frequency of positive alleles of the disease-resistant genes in 119 varieties is 3-10 genes. In japonica rice, 6-9 genes are mainly used, and in indica rice, 4-6 genes are mainly used (B in FIG. 2). Correlation analysis shows that the resistance level is in weak positive correlation with the number of disease-resistant genes (R ² ＝0.0508,P<0.05 (C in fig. 2).

14 disease resistance genes were detected in total in 119 rice main varieties by using 16 SSR molecular markers, and all the disease resistance genes exist in the indica rice and japonica rice subspecies genome (D in FIG. 2). The highest frequency of Pish distribution (100%) and the lowest frequency of Pigm distribution were detected only in Jinxiangyu No. 1 and the 1532 varieties of Anhui rice (Table 4). Furthermore, although the number of polished round-grained nonglutinous rice is much higher (94/25) than that of indica rice, the distribution frequency of other disease-resistant genes is higher in polished round-grained nonglutinous rice than in indica rice (D in FIG. 2 and Table 4). The distribution characteristics of the disease resistance genes indicate that the resistance of indica rice to rice blast may be different.

TABLE 4 distribution of disease resistance genes in 119 Main cultivars of Rice

The "+" indicates the presence; "-" means absent.

EXAMPLE 4 Cluster analysis of 119 Rice major varieties

At the 60% genetic similarity coefficient level, 119 major rice varieties were divided into two clusters (I and II) (fig. 3). The main cluster I contains 107 varieties and is divided into two sub-clusters IA and IB, the sub-cluster IA contains 78 varieties and is divided into two sub-clusters IA-1 and IA-2. 64 varieties were detected in total from IA-1, of which 13 (20.31%) were resistant to disease. IA-2 consisted of 14 varieties of which 7 resistant genotypes (50.00%). The IB sub-population contains 29 varieties, divided into two sub-populations of IB-1 and IB-2, with the majority of indica varieties (21/25) clustered in both sub-populations. The subgroup b-1 consists of 9 varieties, wherein the disease-resistant varieties are 100%. The subgroup b-2 detected a total of 20 varieties, 15 (75.00%) of which were resistant. Interestingly, most (23/29) rice varieties of IB sub-clusters grew in Subei, while rice varieties of other clusters grew in Subei, suzhong and Sunan. Class II is divided into two subgroups IIA (1) and IIB (11), 7 varieties (58.33%) resistant. The results show that the IB subgroup contains most indica rice varieties and has better resistance. Some rice varieties with similar ecological environments belong to the same cluster, and the genetic similar genotypes of all clusters have the characteristics of different ecological environment varieties.

Example 5 disease resistance Gene contribution Rate and disease resistance Gene combination Pattern analysis

The distribution of 14 disease resistance genes in 119 main varieties of rice was analyzed using 16 SSR markers (table 4 and fig. 4). The distribution frequency of the Pish gene was highest (100%), followed by Pit (95.80%) and Pia (80.67%), and the distribution frequency of the Pigm gene was lowest (1.68%), followed by Pi2 (15.18%) and Pi5, piz-t (24.37%). In addition, the resistance contribution rate of each disease resistance gene was calculated based on the resistance comprehensive index. Interestingly, the resistance contribution rate of Pigm was highest (100%), since only two varieties (Jinxiangyu No. 1 and Anhui rice 153) detected Pigm, exhibited resistance to neutralization. The resistance contributions of Pi5, pi2, pita, pikm and Piz-t were 65.52%, 61.11%, 58.49%, 58.23% and 51.72%, respectively, all greater than 50%.

In order to explore which disease resistance genes affect rice resistance, the relationship between the level of resistance and the disease resistance genes was analyzed using multiple stepwise regression. By fitting the model, the 14 variables are input and passed throughThe analysis found that the introduced Pi9, pib, pit, pita, pi5, pigm, pb17 variables were not removed (table 5), indicating that model 7, which contained 7 disease resistance genes, was the best model (R ² ＝0.434,Sig＝0.000)。

TABLE 5 analysis of 7 variables affecting resistance

The above results indicate that Pi9, pib, pit, pita, pi5, pigm and Pb1 have a significant effect on rice resistance. In combination with the drug resistance contribution rate data described above, we analyzed the drug resistance contribution rate of the gene polymerization model (table 6).

TABLE 6 analysis of the contribution rate and distribution frequency of resistance to different gene combinations

A total of 16 combinations of 3 genes, 6 combinations of 4 genes, including 8 disease-resistant genes Pita, pi5, pi9, pib, pb1, pikm, piz-t and Pi2 were found (Table 6). The distribution frequency of the 22 gene combinations was low, ranging from 0.84% (pita+pitm+pi5+piz-t, pita+pitm+pi2+piz-t) to 16.81% (pi9+pib+pb1). In contrast, the resistance contribution rate of these gene combinations is higher, 16.67% -100%. The contribution rate of 6 combined resistances of Pita+Pikm+Piz-t, pita+Pikm+Pi2, pita+Pi5+Piz-t, pikm+Piz-t+Pi2, pita+Pikm+Pi5+Piz-t, pita+Pikm+Pi2+Piz-t reaches 100%. It is notable that there are few varieties containing these gene combinations, only 5 varieties combining Pikm+Piz-t+Pi2, with the remaining 5 combinations being only 1-2 varieties. These results indicate that these key disease-resistant genes or gene combinations have great potential for use in Jiangsu province.

Example 6 detection of Gene combinations in highly resistant Rice varieties

In order to further verify the application of the gene combination in rice disease resistance, 30 rice germplasm resources with better resistance stored in the laboratory are selected, the comprehensive index of the rice resource resistance is above grade 3, meanwhile, 6 gene combinations of 30 rice germplasm resources are analyzed by utilizing molecular markers (according to the operation of the material and method part), and the result shows that 6 gene combinations can be detected, pita+Pi5+Piz-t and Pikm+Piz-t+Pi2 can be detected in 3 rice varieties, and the rest gene combinations are detected in 1 rice variety. In addition, the rice variety SF316 can detect the gene combination in the 6, and has the disease resistance index of 1 and the best resistance.

Table 8 Gene polymerization combinations in 30 resistant rice varieties

The "+" indicates the presence; "-" means absent.

In conclusion, the rice blast germ can infect the whole growth period of the rice, mainly infects leaves and spike parts, and the rice yield is furthest damaged by the spike blast. There have been studies reporting that there is a correlation between the resistance of rice varieties to leaf blast and ear blast, but there are also cases where the resistance is inconsistent. Therefore, the comprehensive evaluation of rice blast resistance by utilizing leaf blast and spike blast is an important technical means for rice disease resistance breeding and variety resistance evaluation. The application evaluates the comprehensive resistance level of 119 main cultivated rice varieties to leaf blast and spike blast in 2019, 2020 and 2021, and the data more objectively and accurately reflect the resistance of 119 varieties to rice blast. The polymerization of the effective resistance gene can effectively improve the resistance of rice varieties. In addition, the broad-spectrum durable resistance of the rice can be better realized through the distribution of resistance genes in the rice variety, the development and the utilization of new resistance genes and the monitoring of non-toxic genes of rice blast bacteria.

The numerical values set forth in these examples do not limit the scope of the present application unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.

Claims

1. A rice blast multiple disease-resistant gene combination is characterized in that the multiple disease-resistant gene combination isPikm+Piz-t+Pi2。

2. The use of the rice blast multiple disease-resistant gene combination according to claim 1,

b) Comparing the resistance of the rice blast to be detected;

3. The use of a reagent for detecting a rice blast multiple disease-resistant gene combination as defined in claim 1 for the preparation of a product having any one of the following functions,

d) Selecting or screening rice varieties with broad-spectrum durable rice blast resistance;

e) Comparing the resistance of the rice blast to be detected;

f) Selecting or screening rice varieties with relatively strong rice blast resistance.

4. The use according to claim 3, wherein the product is a detection kit.

5. In any of the methods described in the following,

(A1) Extracting genome DNA of rice to be detected,

(A2) Performing PCR amplification on genome DNA of rice to be detected, wherein if the genome DNA contains the rice blast multiple disease resistance gene combination of claim 1, the rice to be detected is a rice variety with broad-spectrum durable rice blast resistance;

(B1) Extracting genome DNA of rice to be detected,

(B2) Performing PCR amplification on genome DNA of rice to be detected, wherein the rice blast resistance of the rice variety containing the rice blast multiple disease resistance gene combination of claim 1 is stronger than that of the rice variety containing no rice blast multiple resistance gene combination of claim 1;

(C1) Extracting genome DNA of rice to be detected,

(C2) Performing PCR amplification on genome DNA of rice to be detected, wherein the genome DNA contains the rice blast multiple disease resistance gene combination as set forth in claim 1, so that the rice to be detected is a rice variety with relatively strong rice blast resistance.