阅读说明：本技术 一种在不同植物病害系统中通过病原物的接种即可鉴定抗病基因的技术体系 (Technical system capable of identifying disease-resistant genes by inoculating pathogens in different plant disease systems ) 是由 潘庆华 文健强 张瑞洁 赖琪 王玲 于 2020-06-11 设计创作，主要内容包括：本发明公开了一种在不同植物病害系统中通过病原物的接种即可鉴定抗病基因的技术体系。该方法根据植物病害系统存在的“基因对基因”互作关系,在不同的植物病害系统中鉴定寄主植物抗病基因,具体是从病原物供体菌株中分离克隆其无毒基因并导入受体菌株中而获得重组菌株,将供体菌株、受体菌株及重组菌株分别接种于寄主植物品种上而获得“供体菌株-受体菌株-重组菌株”之“三连反应型”；再通过比较、确认“三连反应型”之间具有的遗传背景的逻辑性及无毒基因的特异性,由此以既具有遗传背景逻辑性又具有无毒基因特异性的“三连反应型”来鉴定与无毒基因互作的抗病基因。本技术体系具有泛用性,可应用于在不同的植物病害系统中发掘、鉴定及导入抗病基因；本技术体系无需分子生物学实验的条件、经验及技术,通过常规的病原物接种方法即可简易而准确地鉴定抗病基因。(The invention discloses a technical system for identifying disease-resistant genes by inoculating pathogens in different plant disease systems. The method identifies host plant disease-resistant genes in different plant disease systems according to the gene-gene interaction relationship existing in the plant disease systems, and specifically comprises the steps of separating and cloning avirulence genes from pathogen donor strains and introducing the avirulence genes into acceptor strains to obtain recombinant strains, and respectively inoculating the donor strains, the acceptor strains and the recombinant strains on host plant varieties to obtain a triple reaction type of 'donor strains-acceptor strains-recombinant strains'; and comparing and confirming the logicality of the genetic background and the specificity of the avirulence gene between the three-linked reaction types, thereby identifying the disease-resistant gene interacting with the avirulence gene by the three-linked reaction type with the logicality of the genetic background and the specificity of the avirulence gene. The technical system has universality and can be applied to the discovery, identification and introduction of disease-resistant genes in different plant disease systems; the technical system can simply and accurately identify the disease-resistant gene by a conventional pathogen inoculation method without the conditions, experience and technology of molecular biology experiments.)

1. A technical system for identifying disease-resistant genes through inoculation of pathogens in different plant disease systems is characterized by comprising the following 3 pathogen test strains with clear genotypes:

(1) donor strain: contains target gene (in particular to avirulence gene AvrX, the same below), the genetic background is donor (donor), and the genotype (target gene/genetic background; the same below) is marked as AvrX/dn;

(2) recipient strain: contains a toxic gene avrX, the genetic background is a receptor (receptor), and the genotype is recorded as avrX/rp;

(3) recombinant strains: the gene contains avirulent gene AvrX, the genetic background is a receptor (receptor), and the genotype is recorded as AvrX/rp;

the recombinant strain is obtained by separating and cloning the avirulence gene from a donor strain and introducing the avirulence gene into a recipient strain;

(4) therefore, the genetic background and the target gene among the test strains can be compared and verified in the technical system so as to identify whether the phenotype of the host plant variety is determined by the target gene of the test strain or the genetic background (other avirulence genes) except the target gene.

2. The technical system of claim 1, wherein the principle and the characteristics are as follows:

(1) respectively inoculating the donor strain, the acceptor strain and the recombinant strain on host plant varieties to obtain a triple reaction type of the donor strain, the acceptor strain and the recombinant strain;

(2) in any 3 strain combinations, all possible "triple reactions" are 8 (2)³8; as R-R-R, S-R-R, R-S-R, R-R-S, R-S-S, S-R-S, S-S-R, S-S-S);

(3) it was concluded from the above-mentioned target genes of the 3 test strains and their genetic backgrounds that none of the 3 "triple connected reactivity types" (R-R-S, S-R-S, S-S-R) exhibited logical properties with genetic backgrounds between the "donor strain-recipient strain-recombinant strain", and thus were practically impossible to exist;

(4) of the 5 actually existing "three-linked reaction types" (R-R-R, S-R-R, R-S-R, R-S-S, S-S-S), 4 (R-R-R, S-R-R, R-S-S, S-S-S) can show logic with genetic background between the "donor strain-acceptor strain-recombinant strain", but do not have the specificity of the target gene, so that the reaction types are actually existing but can not presume the disease-resistant gene corresponding to the target gene;

(5) only "R-S-R" shows both the logical property of the genetic background and the specificity of the target gene between "donor strain-recipient strain-recombinant strain", and thus it can be presumed that the test variety contains a functional disease-resistant gene X corresponding to AvrX.

3. The technical system according to claim 1 or 2, wherein the logical property of having genetic background between the "triple-linked reaction types" means the possibility that the genetic background (including but not limited to other avirulence genes) determines the phenotype of the host plant in addition to the specific avirulence gene AvrX between the 3 test strains.

4. The technical system according to claim 1 or 2, wherein the specificity of the avirulence genes between the "triple-linked reaction types" means the certainty that the phenotype of the host plant is determined by the interaction of the specific avirulence gene AvrX with the corresponding disease resistance gene X between the 3 test strains.

5. The use of the technical system of any one of claims 1 to 4 for the discovery, identification and introduction of disease-resistant genes in different plant disease systems.

6. The use of claim 5, wherein the different phytosanitary systems include, but are not limited to, the rice phytosanitary system.

Technical Field

The invention belongs to the technical field of agricultural biology. More particularly, it relates to a technical system for identifying disease-resistant genes in different plant disease systems.

Background

What is the relationship between host plants and pathogens during the lengthy co-evolution? In this regard, the plant genetics, microbiology and plant pathology kingdoms have been long sought. The famous American scholars of Flolol in the fortieth century suggested a "gene-to-gene" relationship between Fang-for-gene (gene-for-gene; Flor1942, Phytopathology,32: 653) -669) through genetic studies on the resistance to flax rust. That is, in the host plant, a resistance gene (R) and a susceptible allele (R) are present; among pathogens, the avirulence gene (Avr) and the virulence allele (Avr) are present; only when the disease resistance gene interacts with the avirulence gene can the host plant express the disease resistance. The wonderful "gene-to-gene" interaction relationship among the scientists in the multiple disciplines of crop genetics and breeding, plant pathology and microbiology has been long controversial and explored. Until The nineties of The twentieth century, when The isolation and cloning of Plant disease-resistant genes and their corresponding pathogen avirulence genes became possible, The "gene-to-gene" relationship was not considered to be one of The most important relationships between The two after The interaction was demonstrated by protein interaction techniques (Kearney et al 1990, Nature,346: 285-286; Van Kan et al 1991, Molecular Plant-Microbe Interactions,4: 52-59; Meeley et al 1992, Plant Cell,4: 71-77; Martin et al 1993, Science,262: 1432-1436; Mindrinos et al 1994, Cell,78: 1089-1099; Jia et al 2000, The EMBO journal,19: 4004-4014; Keen 2000, nual Review of physiology, 338: 31-48). Thus, this study of Flolol is also considered to be a work in the milestone in the field of plant pathology in the twentieth century, while the "Gene-to-Gene" theory is considered to be one of the core theories for studying the interaction of host plants with pathogens (Valent 1990, Phytopathology,1990,80: 33-36; Keen 2000, Annual Review of Phytopathology,338: 31-48).

Since the theory of "gene-to-gene" and its principle, the identification of disease-resistant genes in plant disease systems can be roughly divided into 2 stages of development:

the first development stage (the forty-eighties of the twentieth century): the molecular biology techniques (including genetic engineering, functional genomics, proteomics, etc.) at this stage have not been fully developed, and people can only advance basic research related to disease-resistant genes and avirulence genes by constructing a differentiation system of plant disease systems (Lingzhi et al, 2004, Chinese agricultural science, 37: 1849-. The term "variety discrimination" means a set of representative varieties having different disease-resistant genotypes to classify the pathotype (physiological races) of a test strain according to the response type thereof; the identification of strains refers to a set of representative strains having different avirulence genotypes to classify the disease-resistant genotypes of the test varieties according to their reactivity (Atkins et al 1967, Phytopathology 57: 297-.

The rice blast model disease system will be described as an example. The rice blast scholars who take the Qingzemaotai as the first thing select 7 representative strains as the identification strains of the Japanese rice blast system by utilizing the survey data of the Japanese national rice blast physiological race cooperation group for 7 years; then, the set of identifying strains is used for classifying disease-resistant genotypes (12 types) of more than 3000 rice varieties in the China; then selecting representative varieties from each type, and identifying 12 disease-resistant genes by means of conventional separation analysis of genetic filial generations and the like; finally, 9 representative varieties (which are upgraded to 12 later) with different disease-resistant genotypes are selected as the discriminators of the Japanese Rice blast system (Qingze Mao Jiu 1974, report of agricultural research, D1, pp 1-58; Qingze Mao Jiu and AnDong Yun Man 1990, Daohuacheng volume III (Genistian Ed.), pp 361-385; Zhang et al.2017, Rice,10: 46). As of the eighties of the twenty century, 14 Rice blast Disease-resistant genes at 8 sites were identified by Qingze Miao and collaborators thereof, which became antecedents and models of the genetic research of the whole Plant Disease system (Qingze Miao and Andong Yun Man 1990, Daohuang Dai third volume (genetic code), pp 361-385; Zhang et al 2017, Rice 10: 46; Zhang et al 2019, Plant Disease 103: 2759) 2763).

The technical limitations at this stage are: (1) the disease-resistant genotypes of a large number of varieties can only be classified by using a discrimination system; (2) the identification of disease-resistant genes relies mainly on conventional genetic segregation analysis, and few involve linkage analysis based on morphological trait markers.

The second stage of development (eighties of the twentieth century-present): at this stage, molecular biology techniques, particularly molecular marker techniques, functional genomics techniques, and other fields have substantially advanced, and gene cloning techniques and sequencing techniques have gradually become routine techniques in common laboratories. The research of disease-resistant genes enters a brand-new era. Therefore, in the case where the genetic rules of many disease-resistant genes have been clarified and located, the cloning and functional studies of disease-resistant genes based on the whole genome sequence of plants and the massive biological information generated therefrom have been fully developed. On one hand, The number of located and discovered disease-resistant genes (containing alleles) in this period is continuously increased, and on The other hand, The number of clones of The disease-resistant genes in this period is also greatly increased (International Rice Genome Sequencing Project 2005, Nature,436: 793-.

The rice blast model disease system will be described as an example. Compared with the first development stage, the positioned disease-resistant genes greatly increase to nearly 100 from 4 (Pis, Pita/Pita-2, Pia, Piz/Piz-t); the number of cloned disease-resistant genes greatly increased from 0 to more than 30 (Qingze Mao Jiu 1974, agricultural Research report, D1, pp 1-58; Sharma et al 2012, Agriculture Research,1: 37-52; Liu and Wang 2016, National Science Review,3: 295-308; Kalia et al 2019,3Biotech,9: 209). On the basis, the efficiency and accuracy of disease-resistant gene discovery, identification and introduction are greatly improved by developing and utilizing a linked marker (aiming at an unclosed gene) or a function-specific marker (aiming at a cloned gene) (ZHai et al 2011, New Phytolist 189: 321-.

The technical limitations at this stage are: although the application of molecular marker technology such as disease-resistant gene linkage marker or function specificity marker improves the efficiency and accuracy of the discovery, identification and introduction of disease-resistant gene. However, the technical operation is relatively complex and has high requirements on operation researchers; for the field production practice, a set of conventional technical system which is simpler, more accurate and independent of molecular markers is still required to be developed to promote the discovery, identification and introduction of disease-resistant genes.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing disease-resistant gene excavation and identification technology, and the primary purpose is to provide a conventional technical system which is simpler, easier and more accurate and does not depend on molecular markers for identifying the disease-resistant genes. And the thinking and the principle of the technical system and the application example of identifying the disease-resistant gene by using the technical system are shown and verified.

The second purpose of the invention is to provide the application of the technical system in the discovery, identification and introduction of disease-resistant genes in different plant disease systems.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a technical system for identifying disease-resistant genes by inoculating pathogens in different plant disease systems, which comprises the following 3 pathogen test strains with clear genotypes:

(1) donor strain: contains target gene (in particular to avirulence gene AvrX, the same below), the genetic background is donor (donor), and the genotype (target gene/genetic background; the same below) is marked as AvrX/dn;

(2) recipient strain: contains a toxic gene avrX, the genetic background is a receptor (receptor), and the genotype is recorded as avrX/rp;

(3) recombinant strains: the gene contains avirulent gene AvrX, the genetic background is a receptor (receptor), and the genotype is recorded as AvrX/rp;

the recombinant strain is obtained by separating and cloning the avirulence gene from a donor strain and introducing the avirulence gene into a recipient strain;

(4) therefore, the genetic background and the target gene among the test strains can be compared and verified in the technical system so as to identify whether the phenotype of the host plant variety is determined by the target gene of the test strain or the genetic background (other avirulence genes) except the target gene.

The principle and the characteristics of the technical system are as follows:

(1) respectively inoculating the donor strain, the acceptor strain and the recombinant strain on host plant varieties to obtain a triple reaction type of the donor strain, the acceptor strain and the recombinant strain;

(2) in any 3 strain combinations, all possible "triple reactions" are 8 (2)³8; is marked as R-R-R, S-R-R, R-S-R, R-R-S, R-S-S, S-R-S, S-S-R, S-S-S; wherein, R, resistance/disease resistance; s, susceptable/susceptibility; the same applies below);

(3) it was concluded from the above-mentioned target genes of the 3 test strains and their genetic backgrounds that none of the 3 "triple connected reactivity types" (R-R-S, S-R-S, S-S-R) exhibited logical properties with genetic backgrounds between the "donor strain-recipient strain-recombinant strain", and thus were practically impossible to exist;

(4) of the 5 actually existing "three-linked reaction types" (R-R-R, S-R-R, R-S-R, R-S-S, S-S-S), 4 (R-R-R, S-R-R, R-S-S, S-S-S) can show logic with genetic background between the "donor strain-acceptor strain-recombinant strain", but do not have the specificity of the target gene, so that the reaction types are actually existing but can not presume the disease-resistant gene corresponding to the target gene;

(5) only "R-S-R" shows both the logical property of the genetic background and the specificity of the target gene between "donor strain-recipient strain-recombinant strain", and thus it can be presumed that the test variety contains a functional disease-resistant gene X corresponding to AvrX.

Further, in the above technical system of the present invention, the logical property of having genetic background between the "triple tandem reaction type" means the possibility that the genetic background (including but not limited to other avirulence genes) determines the phenotype of the host plant in addition to the specific avirulence gene AvrX between the 3 test strains.

Further, in the above technical system of the present invention, the specificity of the avirulence gene between the "triple-linked reaction types" means that the necessity of the host plant phenotype is determined by the interaction between the specific avirulence gene AvrX and the corresponding disease resistance gene X among the 3 test strains.

Meanwhile, the application of the technical system in the aspects of discovery, identification, introduction and the like of disease-resistant genes in different plant disease systems is all within the protection scope of the invention.

Wherein, the plant disease system comprises pathogenic bacteria and host varieties thereof. Specifically, the different phytosanitary systems include, but are not limited to, the rice disease system.

Based on the technical system provided by the invention, specifically:

the present invention relates to the hypothesis of the "gene-for-gene" interaction found in the plant disease system (Flor1942, Phytopathology,32:653-669) (FIG. 2). Wherein the content of the first and second substances,

in host plants, there are disease resistance genes and their susceptibility alleles, generally, the disease resistance genes are dominant to the susceptibility genes;

in the pathogen, there is a avirulence gene and its toxic allele, generally a avirulence gene is dominant over a toxic gene;

only when the disease-resistant gene interacts with the avirulence gene can the host plant express the disease resistance;

conversely, it is concluded that, in any plant disease system, as long as the host plant expresses disease resistance, the disease resistance gene of the host plant is functional with the avirulence gene of the pathogen, and that both interact directly or indirectly;

in particular, under the condition that the disease-resistant gene is recessive to the disease-sensitive gene or the avirulent gene is recessive to the toxic gene, the gene-to-gene interaction relationship between the disease-resistant gene and the toxic gene has not been discovered and verified so far, and the cases are excluded from the application patent.

The invention provides a principle and a technical system for identifying disease-resistant genes by using avirulence genes in a plant disease system based on the gene-gene interaction relationship (figure 3). Wherein the content of the first and second substances,

(1) suppose that 8 host plant varieties (respectively containing 8 different disease-resistant genes I to VIII) participate in the test. Wherein, the disease-resistant gene III and the avirulence gene AvrIII have the specific interaction of 'gene to gene';

(2) it was assumed that 3 pathogen strains participated in the experiment. Wherein the content of the first and second substances,

donor strain: contains target gene (in particular to avirulence gene AvrIII, the same below), the genetic background is donor (donor), and the genotype (target gene/genetic background; the same below) is marked as AvrIII/dn;

recipient strain: contains a toxic gene avrIII, the genetic background is a receptor (receptor), and the genotype is recorded as avrIII/rp;

recombinant strains: contains avirulent gene AvrIII, the genetic background is receptor (receptor), and the genotype is recorded as AvrIII/rp.

Therefore, the target gene and genetic background are different between the "donor strain and the recipient strain"; between the donor strain and the recombinant strain, the target genes are the same, but the genetic background is different; however, the target gene is not the same but the genetic background is the same between the "recipient strain and the recombinant strain". Therefore, the genetic background and the target gene among the test strains can be compared and verified in the technical system so as to distinguish whether the phenotype of the host plant variety is determined by the target gene of the test strain or the genetic background (including but not limited to other non-toxic genes) except the target gene.

(3) In any 3 combinations of strains [ take the example of "donor strain-acceptor strain-recombinant strain", the order between the three does not affect the logical relationship and reasoning results, the same below ], and all possible "triple reaction types" are 8 (2)³8; R-R-R, S-R-R, R-S-R, R-R-S, R-S-S, S-R-S, S-S-R, S-S-S); therefore, for 8 test varieties, each variety corresponds to 1 'three-linking reaction type';

however, it was concluded from the above-mentioned 3 test strains that the genes of interest and their genetic backgrounds were such that none of the 3 "triple connected responses" (R-R-S, S-R-S, S-S-R) exhibited logical properties with genetic backgrounds between the "donor strain-recipient strain-recombinant strain" and were therefore responses that were virtually impossible to exist.

Further, of the 5 actually existing "triple-linked reaction types" (R-R-R, S-R-R, R-S-R, R-S-S, S-S-S), 4 of them (R-R-R, S-R-R, R-S-S, S-S-S) showed logical properties with genetic background between "donor strain-recipient strain-recombinant strain", but did not have specificity of the objective gene, so these reaction types were actually existing but could not presume the corresponding disease-resistant gene of the objective gene;

only "R-S-R" shows both the logical property of the genetic background and the specificity of the target gene between "donor strain-recipient strain-recombinant strain", and thus it can be presumed that the test variety III contains the functional disease-resistant gene III corresponding to AvrIII.

In summary, the principle and technical system of the invention are as follows:

(a) the avirulence gene is a necessary condition for identifying the disease-resistant gene;

(b) the 'triple reaction type' which is originated from the 'donor strain, the receptor strain and the recombinant strain' and has the logical genetic background is a sufficient condition for identifying the interactive disease-resistant genes;

(c) the 'triple reaction type' which is originated from the 'donor strain, the receptor strain and the recombinant strain' and has both genetic background logicality and target gene specificity is a sufficient necessary condition for identifying the interacting disease-resistant genes.

The present invention provides an example of identifying a corresponding disease-resistant gene Pit based on a "gene-to-gene" interaction with the avirulence gene AvrPit of Pyricularia oryzae (FIG. 4). Wherein the content of the first and second substances,

(1) it was assumed that 5 rice varieties (each containing 5 different disease resistance genes) participated in the experiment. Wherein, the avirulence gene AvrPit and the disease-resistant gene Pit have the specific interaction of 'gene to gene';

(2) it was assumed that 3 rice blast strains participated in the experiment. Wherein the content of the first and second substances,

donor strain [ CHL 357; contains avirulent gene AvrPit, the genetic background is donor (donor), and the genotype is recorded as AvrPit/dn';

recipient strain [ Guy 11; contains a toxic gene avrPit, the genetic background is a receptor (receptor), and the genotype is recorded as avrPit/rp;

recombinant strains [ At 13-9; contains avirulence gene AvrPit, the genetic background is receptor (recipient), and the genotype is recorded as AvrPit/rp.

(3) Of the 5 actually existing "triple-tandem reaction types", only "R-S-R" can presume that the test variety K59 contains a disease-resistant gene Pit corresponding to Avrpit, and the remaining 4 test varieties do not contain a target gene, i.e., other disease-resistant genes.

(4) The above results were obtained by using a specific molecular marker Pit for a disease-resistant gene^A2338GThereby obtaining the authentication.

The present invention provides a comparative example of the principle of identifying disease-resistant genes by using an identification system based on the "gene-to-gene" interaction relationship in a plant disease system (FIG. 5). Wherein the content of the first and second substances,

(1) the test is assumed to be participated by 7 host identification varieties (respectively containing 7 different disease-resistant genotypes I-VII) and 3 pathogen identification strains (respectively containing different avirulence genotypes but unknown genetic background and target genes);

(2) the number of the triple reaction type generated by the interaction of 7 identification varieties and 3 identification strains and having at least 1 disease-resistant reaction is 7 (R-R-R, S-R-R, R-S-R, R-R-S, R-S-S, S-R-S, S-S-R-R).

(3) Further assuming that the disease-resistant genes (types) have additive effects, the disease-resistant genes (types) presumed from the above 7 "triple-linked reaction types" are as follows:

the possible disease-resistant genes (types) are presumed to be 14 from the "R-R-R", and thus any specific disease-resistant gene (type) cannot be presumed;

the possible disease-resistant genes (types) are presumed to be 4 by the "S-R-R", "R-S-R" or "R-R-S", so that any specific disease-resistant gene (type) cannot be presumed;

the number of possible disease-resistant genes (types) estimated by "R-S-S", "S-R-S" or "S-S-R" is only 1, although it is possible to estimate the respective disease-resistant gene types corresponding to the varieties to be discriminated. However, since the objective gene (avirulence gene) and genetic background between strains to be identified are unknown, a specific disease-resistant gene cannot be finally confirmed.

In conclusion, since the identification of target genes and genetic backgrounds among strains are unknown, the technical system can be applied to classification of disease-resistant genotypes (all 29 types) of host plant varieties, but cannot be applied to identification of specific disease-resistant genes.

The present invention provides comparative examples for identifying disease resistance genes in a plant disease system using disease resistance gene-specific molecular markers (FIG. 6). Wherein the content of the first and second substances,

(1) 2 specific molecular markers are developed according to the sequence and structure comparison difference of rice blast Pib resistance/sensitivity alleles;

(2)1 control variety (CK: IRBLb-B, carrying Pib), 12 Japanese cultivars (Shin2, AichiAsahi, Fujisaka 5, Kusabue, Tsuyuake, Fukunsihiki, K1, PiNo.4, Toride1, K60, BL1, K59), and 1 common cultivar (CO39) were tested;

(3) marker Pib-1^P/AThe detection result shows that the control variety IRBLb-B and 2 test varieties (BL1 and CO39) contain the target gene;

(4) marker Pibdom^P/ADisplay of the results of the detectionControl variety IRBLb-B and 1 test variety (BL1) contained the gene of interest, whereas CO39 did not;

in fact, only the control variety IRBLb-B and the test variety BL1 contain the target gene. Thus, Pib-1^P/AEven if the molecular marker is gene-specific, the result of the identification is false positive.

The principle and the technical system for simply and accurately identifying the disease-resistant genes in different plant disease systems provided by the invention have important application values: by utilizing the principle and the technical system and through a conventional pathogen inoculation method, the application of discovering, identifying and introducing disease-resistant genes in different plant disease systems is realized.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a technical system for identifying disease-resistant genes in different plant disease systems, which comprises the following steps:

(1) the invention has general application, is not only suitable for rice blast disease systems, but also can be used for any plant disease system, and can be applied to the discovery, identification and introduction of disease-resistant genes in different plant disease systems;

(2) the invention can simply and accurately identify the disease-resistant gene by a conventional pathogen inoculation method without the conditions, experience and technology of molecular biology experiments;

(3) compared with the identification technology of an identification system, the invention can not only separate the disease-resistant genotypes of host plant varieties, but also accurately identify functional disease-resistant genes;

(4) compared with the molecular marker identification technology, the invention can accurately identify the functional disease-resistant gene and avoid the occurrence of non-functional disease-resistant gene (false positive marker).

Drawings

FIG. 1 is a technical route chart for identifying disease-resistant genes in a plant disease system easily and accurately.

FIG. 2. Gene-to-gene interactions present in the plant disease system; wherein the content of the first and second substances,

in the host plant, there is a disease resistance gene (R) and its susceptibility allele (R), generally the resistance gene is dominant to the susceptibility gene;

in the pathogen, there is an avirulence gene (Avr) and its virulence allele (Avr), generally a avirulence gene is dominant over a virulence gene;

only when the disease-resistant gene interacts with the avirulence gene can the host plant express the disease resistance;

conversely, it is concluded that, in any plant disease system, as long as the host plant expresses disease resistance, the disease resistance gene of the host plant is functional with the avirulence gene of the pathogen, and that both interact directly or indirectly;

in particular, under the condition that the disease-resistant gene is recessive to the disease-sensitive gene or the avirulent gene is recessive to the toxic gene, the gene-to-gene interaction relationship between the disease-resistant gene and the toxic gene has not been discovered and verified so far, and the cases are excluded from the application patent.

R, resistance gene; r, allele of resistance gene (susceptible allele of disease resistance gene);

avr, avirulence gene (avirulence gene); avr, allele of avirulence gene (virulence allele of avirulence gene);

r, resistance; s, susceptable (susceptibility).

FIG. 3 shows the principle and technical system of identifying disease-resistant genes by non-toxic genes in a plant disease system based on the gene-to-gene interaction relationship;

FIG. 3 a: assuming that 8 host plant varieties (respectively containing 8 different disease-resistant genes I-VIII) participate in the test, wherein the disease-resistant gene III and the avirulence gene AvrIII have the specific interaction of 'gene to gene';

FIG. 3 b: the donor strain [ contains avirulence gene AvrIII, the genetic background is a donor (donor), and the genotype is recorded as the reaction type of AvrIII/dn ] to 8 test varieties;

FIG. 3 c: the recipient strain contains a toxic gene avrIII, the genetic background is a recipient (recipient), and the genotype of the recipient strain is recorded as the reaction type of the avrIII/rp to 8 test varieties;

FIG. 3 d: the recombinant strain [ contains avirulence gene AvrIII, the genetic background is a receptor (recipient), and the genotype is recorded as the reaction type of AvrIII/rp ] to 8 test varieties;

FIGS. 3 e-f: in any 3 strain combinations [ take "donor strain-recipient strain-recombinant strain" as an example ], all possible "triple reactions" are 8 (2)³8); therefore, for 8 test varieties, each variety corresponds to 1 'three-linking reaction type'; however, it was concluded from the above-mentioned 3 test strains that the genes of interest and their genetic backgrounds were such that none of the 3 "triple connected responses" (R-R-S, S-R-S, S-S-R) exhibited logical properties with genetic backgrounds between the "donor strain-recipient strain-recombinant strain" and were therefore responses that were virtually impossible to exist. Further, of 5 actually existing "triple reaction types" (based on which classification of disease-resistant genotypes of host varieties can be made), 4 (R-R-R, S-R-R, R-S-S, S-S-S) among them all can exhibit logic with genetic background between "donor strain-recipient strain-recombinant strain", but do not have specificity of the target gene, and thus these reaction types are disease-resistant genes that may actually exist but cannot presume the target gene corresponds to; only "R-S-R" shows both the logical property of the genetic background and the specificity of the target gene between "donor strain-recipient strain-recombinant strain", and thus it is presumed that the test variety III contains a functional disease-resistant gene III corresponding to AvrIII.

R, resistance; s, susceptable (susceptibility).

FIG. 4 is a diagram identifying an example of a corresponding disease-resistant gene Pit based on a "gene-to-gene" interaction with the avirulence gene Avrpit of Pyricularia oryzae;

FIG. 4 a: 5 rice varieties (respectively containing 5 different disease-resistant genes) are assumed to participate in the test;

FIGS. 4 b-c: donor strain [ CHL 357; the gene contains an avirulent gene AvrPit, the genetic background is a donor (donor), and the genotype is recorded as the reaction type of AvrPit/dn) to 5 test varieties;

FIGS. 4 d-e: recipient strain [ Guy 11; contains a toxic gene avrPit, the genetic background is a receptor (receptor), and the genotype is recorded as the reaction type of the avrPit/rp to 5 test varieties;

FIGS. 4 f-g: recombinant strains [ At 13-9; the gene contains an avirulent gene AvrPit, the genetic background is a receptor (receptor), and the genotype is recorded as the reaction type of AvrPit/rp to 5 test varieties;

of the 5 actually existing "triple-tandem reaction types", only "R-S-R" can presume that the test variety K59 contains a disease-resistant gene Pit corresponding to Avrpit, and the remaining 4 test varieties do not contain the disease-resistant gene, that is, contain other disease-resistant genes.

FIG. 4 h: specific molecular marker Pit using disease-resistant gene^A2338GIdentification and verification of disease resistance gene Pit. The left side is the DNA molecular weight marker, DNA ladder-500 bp.

R, resistance; s, susceptable (susceptibility).

FIG. 5 shows a comparative example of the principle of identifying a disease-resistant gene using an identification system based on the "gene-to-gene" interaction in a plant disease system;

FIGS. 5 a-d: the test is supposed to be participated by 7 host plant identification varieties (respectively containing 7 different disease-resistant genes I-VII) and 3 pathogen identification strains (respectively containing different avirulence genotypes but unknown genetic background and target genes); thus, the three-linked reaction type generated by the interaction of 7 varieties and 3 strains is 7 (R-R-R, S-R-R, R-S-R, R-R-S, R-S-S, S-R-S, S-S-R) with at least 1 disease-resistant reaction;

FIG. 5 e: further assuming that the disease-resistant genes (types) have additive effects, the disease-resistant genes (types) presumed from the above 7 "triple-linked reaction types" are as follows:

the possible disease-resistant genes (types) are presumed to be 14 from the "R-R-R", and thus any specific disease-resistant gene (type) cannot be presumed;

the possible disease-resistant genes (types) are presumed to be 4 by the "S-R-R", "R-S-R" or "R-R-S", so that any specific disease-resistant gene (type) cannot be presumed;

since only 1 possible disease-resistant gene (type) is estimated from "R-S-S", "S-R-S" or "S-S-R", although it can be estimated to correspond to each disease-resistant genotype of the variety to be identified, it is impossible to finally confirm a specific disease-resistant gene because the target gene (avirulence gene) and genetic background between strains to be identified are unknown. That is, any disease-resistant gene in the identification variety disease-resistant genotype can control the disease-resistant response.

In conclusion, since the identification of target genes and genetic backgrounds among strains are unknown, the technical system can be applied to the classification of disease-resistant genotypes (all 29 types) of host plant varieties, but cannot be applied to the identification of specific disease-resistant genes.

R, resistance; s, susceptable (susceptibility).

FIG. 6 comparative examples of identification of disease resistance genes based on disease resistance gene-specific molecular markers in a plant disease system;

FIG. 6a is a diagram showing the comparison of the sequence and structure of the Pib resistance/sensitivity allele and the development of its specific molecular marker;

FIG. 6B 1 control cultivars (CK: Pib, IRBLb-B), 12 Japanese cultivars (Shin2, Aichi Asahi, Fujisaka 5, Kusabue, Tsuyuake, Fukunishiki, K1, PiNo.4, Toride1, K60, BL1, K59), and 1 common cultivar (CO39) were tested;

FIG. 6c shows that the disease-resistant gene Pib-specific molecular marker Pib-1^P/AThe electrophoresis pattern of the PCR amplification product. The results show that the control variety IRBLb-B and the test varieties BL1 and CO39 both contain Pib;

FIG. 6d shows that the disease-resistant gene Pib specific molecular marker Pibdom^P/AThe electrophoresis pattern of the PCR amplification product. The results show that the control variety IRBLb-B and the test variety BL1 contain Pib, whereas CO39 does not contain Pib.

In fact, only the control variety IRBLb-B and the test variety BL1 contain the target gene. Thus, the mark Pib-1^P/AEven if the molecular marker is gene-specific, the result of the identification is false positive.

In conclusion, due to the diversity of the sequence and structural differences of the anti-sense/susceptibility alleles, even gene-specific molecular markers are inevitably subject to false positive identification.

M, DNA molecular weight marker, DNA ladder-2000 bp.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the embodiments of the present invention include, but are not limited to, the following examples. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

In the examples section of the present invention, the "gene-to-gene" interactions that exist in plant disease systems are illustrated; the principle and the technical system of identifying the disease-resistant genes by using the avirulence genes are used in a plant disease system based on the gene-to-gene interaction relationship; identifying the corresponding examples of the disease-resistant gene Pit based on the gene-to-gene interaction relation with the avirulence gene Avrpit of the rice blast germs; a principle comparison example for identifying disease-resistant genes by using an identification system based on a gene-to-gene interaction relationship in a plant disease system; and a comparative example of identifying a disease-resistant gene using a disease-resistant gene-specific molecular marker in a plant disease system. The technical route adopted by the invention is shown in figure 1.

The donor strain CHL357 of AvrPit used in the examples, as well as the recipient strain Guy11(Zeng et al 2009, Plant Disease,93: 238-; the rice varieties used were: IRBLzt-T, IRBLb-B, IRBLz5-CA, IRBLi-F5, IRBLt-K59, and Shin2, Aichi Asahi, Fujisaka 5, Kusabue, Tsuyuake, Fukunishihiki, K1, PiNo.4, Toride1, K60, BL1, K59, CO39(Table S3 inZhai et al 2011, New Phytolist, 189: 321-; the specific molecular markers of the rice blast disease-resistant genes are as follows: pit^A2338G,Pib-1^P/A,Pibdom^P/A(Lai Qi 2019, Master academic paper of university of agriculture in south China; Robert et al 2004, Crop Science,44: 1790-; recombinant strain of AvrPitAt13-9 was constructed by the inventors by isolating and cloning AvrPit and then introducing it into the recipient strain Guy11, and was disclosed in the patent of "Magnaporthe oryzae avirulence gene AvrPit and its use" filed by the inventors (201911024985.0).