Specific molecular marker primer set for rice blast resistance gene and its application

Abstract

The present invention relates to the field of molecular genetic breeding of crops, and specifically to a rice blast resistance gene-specific molecular marker primer set and its application. After multiple positive and negative controls or resequencing tests of partial results, effective molecular markers are developed for eight resistance genes, namely, Pit, Pish, Pib, Pid3, Pi5, Pia, Pi54 and Pita2 / Ptr. Through molecular marker identification, the composition of resistance genes in parents in the Sichuan Basin is revealed, the application of parents in resistance breeding is promoted, and the efficiency of resistance gene infiltration and polymerization is improved through molecular assisted breeding. In addition, the composition of resistance genes can be clarified according to the incidence of each variety in rice production, so as to reveal the disease resistance of each resistance gene or resistance gene combination in different regions, and provide guidance for green prevention and control of rice disease resistance, variety layout and disease resistance breeding.

Description

Technical Field

[0001] The invention relates to the field of crop molecular genetic breeding, and in particular to a rice blast resistance gene specific molecular marker primer set and application thereof. Background Art

[0002] Breeding resistant varieties is the most economical, environmentally friendly and effective way to improve the resistance of rice varieties. At present, more than 50 rice blast resistance genes have been cloned from rice, distributed in 17 resistance gene loci. According to the identification results of rice blast resistance in various rice-growing areas across the country, single-gene lines containing Pi2 or Pik locus resistance genes, such as Pi2, Pi9 or Pikm, have good broad-spectrum resistance. However, with the widespread application of resistance genes in rice resistance breeding, single resistance genes gradually lose resistance. However, the aggregation of different resistance genes generally has the effect of improving rice blast resistance. For example, rice materials containing PigmR+Pi1, PigmR+Pi54 or PigmR+Pi33 resistance genotypes have stronger resistance to rice blast than rice materials containing a single resistance gene. Resistance gene combinations Pib+Pi9, Pib+Pi2, Pib+Pish and Pi9+Pi54 also show good resistance to rice blast. In some rice varieties, the more resistance genes are aggregated, the stronger the resistance to rice blast. Therefore, the aggregation of Pi2 or Pik resistance genes with other resistance genes, or the aggregation of resistance genes at other sites, also has good resistance to rice blast.

[0003] Compared with Pi2 or Pik locus resistance genes, there are fewer molecular markers developed for other resistance genes such as Pit, Pia and Pib, and they are subject to problems such as genetic background and specificity, making it difficult to accurately identify these resistance genes. However, efficient resistance gene aggregation requires specific molecular markers to clarify the resistance gene composition of rice materials, so as to accurately utilize resistance resources, and also requires molecular markers for auxiliary screening to improve the efficiency of resistance breeding and save costs. Therefore, the present invention screens specific nucleotide sites in the coding regions of eight resistance genes (Pit, Pish, Pib, Pid3, Pi5-1, Pia, Pi54 and Pita2 / Ptr) to develop molecular markers, and through multiple positive and negative control materials, and sequencing verification, a set of specific molecular markers is screened for these eight genes. At the same time, the resistance gene composition of 109 breeding parents is accurately and efficiently revealed, resistance gene resources are clarified for resistance breeding, and reliable molecular markers are provided for auxiliary breeding. Summary of the invention

[0004] The object of the present invention is to provide a rice blast resistance gene-specific molecular marker primer set and its application. After multiple positive and negative controls or resequencing tests of partial results, effective molecular markers are developed for eight resistance genes, namely, Pit, Pish, Pib, Pid3, Pi5, Pia, Pi54 and Pita2 / Ptr. Through molecular marker identification, the composition of resistance genes in parents in the Sichuan Basin is revealed, the application of parents in resistance breeding is promoted, and the efficiency of resistance gene infiltration and polymerization is improved through molecular assisted breeding. In addition, according to the incidence of each variety in rice production, its resistance gene composition can be clarified, and the disease resistance of each resistance gene or resistance gene combination in different regions can be revealed, so as to provide guidance for green prevention and control of rice disease resistance, variety layout and disease resistance breeding.

[0005] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0006] Rice blast resistance gene specific molecular marker primer set, including:

[0007] The forward primer is Pit-pF, and the reverse primer is Pit-pR, and their nucleotide sequences are shown in SEQ ID NO.1-2.

[0008] The forward primer is Pit-mF, and the reverse primer is Pit-mR, and their nucleotide sequences are shown in SEQ ID NO.3-4.

[0009] The forward primer is Pish / Pi37-F, and the reverse primer is Pish / Pi35-R, and their nucleotide sequences are shown in SEQ ID NO.5-6.

[0010] The forward primer is Pib-F2, and the reverse primer is Pib-R2, and their nucleotide sequences are shown in SEQ ID NO.7-8.

[0011] The forward primer is Pib-mF, and the reverse primer is Pib-mR, and their nucleotide sequences are shown in SEQ ID NO.9-10.

[0012] The forward primer is Pid3-F2, and the reverse primer is Pid3-R2, and their nucleotide sequences are shown in SEQ ID NO.11-12.

[0013] The forward primer is Pid3-mF, ​​and the reverse primer is Pid3-mR, and their nucleotide sequences are shown in SEQ ID NO.13-14.

[0014] The forward primer is Pi5-1-gF, and the reverse primer is Pi5-1-gR, and their nucleotide sequences are shown in SEQ ID NO.15-16.

[0015] The forward primer is Pi5-1-2F, and the reverse primer is Pi5-1-2R, and their nucleotide sequences are shown in SEQ ID NO.17-18.

[0016] The forward primer is Pia(RGA5)-F, and the reverse primer is Pia(RGA5)-R, and their nucleotide sequences are shown in SEQ ID NO.19-20.

[0017] The forward primer is Pi54-F, and the reverse primer is Pi54-R, and their nucleotide sequences are shown in SEQ ID NO.21-22.

[0018] The forward primer is Pi54-mF, and the reverse primer is Pi54-mR2, and their nucleotide sequences are shown in SEQ ID NO.23-24.

[0019] The forward primer is Ptr / Pi-ta2-F, and the reverse primer is Ptr / Pi-ta2-R, and their nucleotide sequences are shown in SEQ ID NO.25-26.

[0020] The forward primer is Ptr / Pi-ta2-F, and the reverse primer is Ptr-Rs, and their nucleotide sequences are shown in SEQ ID NOs.25 and 27.

[0021] On the other hand, the present invention provides a method for detecting rice blast resistance alleles based on the above primer pairs, comprising the following steps:

[0022] S1: Extract genomic DNA of rice samples to be tested;

[0023] S2: performing PCR amplification using the primer pair described in claim 1;

[0024] S3: Detection of Pit gene based on primer pair Pit-pF, Pit-pR and primer pair Pit-mF, Pit-mR;

[0025] S4: Detection of the Pish gene based on primer pairs Pish / Pi37-F and Pish / Pi35-R;

[0026] S5: Detection of Pib gene based on primer pair Pib-F2, Pib-R2 and primer pair Pib-mF, Pib-mR;

[0027] S6: Detection of Pid3 gene based on primer pair Pid3-F2, Pid3-R2 and primer pair Pid3-mF, ​​Pid3-mR;

[0028] S7: Detection of Pi5 gene based on primer pair Pi5-1-gF, Pi5-1-gR and primer pair Pi5-1-2F, Pi5-1-2R;

[0029] S8: Detection of Pia gene based on primer pairs Pia(RGA5)-F and Pia(RGA5)-R;

[0030] S9: Detection of Pi54 gene based on primer pair Pi54-F, Pi54-R and primer pair Pi54-mF, Pi54-mR2;

[0031] S10: Detection of Ptr / Pi-ta2 gene based on primer pairs Ptr / Pi-ta2-F, Ptr / Pi-ta2-R and primer pairs Ptr / Pi-ta2-F, Ptr-Rs.

[0032] Furthermore, the rice DNA extraction in step S1 specifically includes: 100 mg of rice leaves are placed in a 1.5 mL EP tube, after adding liquid nitrogen and grinding, 500 μl of CTAB solution is added, and the mixture is placed in a 65°C oven for 30 minutes, and mixed every 10 minutes. After cooling to room temperature, 500 μl of chloroform / isoamyl alcohol (24:1) solution is added, vortexed and mixed, and then allowed to stand at room temperature for 5 minutes. Then centrifuge (12000 r / min) for 15 minutes; take the supernatant into a new 1.5 mL EP tube, add an equal volume of anhydrous ethanol, mix and stand in a -20°C refrigerator for 10 minutes. Centrifuge (12000 r / min) for 10 minutes, discard the supernatant, wash the precipitate twice with 1000 ml of pre-cooled 75% anhydrous ethanol, add 100 μl of ddH2O to dissolve the DNA after the precipitate is dried, and store in a -20°C refrigerator for use.

[0033] Furthermore, the PCR reaction system for PCR amplification in step S2 is: 2.0 μL template DNA (400 ng / μL), 2.0 μL Buffer, 0.5 μL dNTP (2.5 mM each), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 0.5 μL Taq enzyme (2.5 U / μL), 14.0 μL ddH2O, a total of 20 μL.

[0034] The invention also discloses the application of the method in rice material gene typing and rice blast resistance breeding.

[0035] The invention also discloses a kit containing the primer pair.

[0036] The invention also discloses the application of the kit in rice material gene typing and rice blast resistance breeding.

[0037] Beneficial effects of the present invention:

[0038] The resistance gene composition of many rice parents is not clear. In order to better apply it to rice resistance breeding, the present invention provides a rice blast resistance gene-specific molecular marker primer set and its application, which is not limited by genetic background, clarifies the resistance gene composition of parents and provides markers for molecular assisted breeding. Through molecular marker identification, the resistance gene composition in parents in the Sichuan Basin is revealed, the application of parents in resistance breeding is promoted, and the efficiency of resistance gene infiltration and polymerization is improved through molecular assisted breeding. In addition, according to the incidence of each variety in rice production, its resistance gene composition can be clarified, and the disease resistance of each resistance gene or resistance gene combination in different regions can be revealed, which provides guidance for green prevention and control of rice disease resistance, variety layout and disease resistance breeding.

[0039] The present invention explores the nucleotide polymorphism sites of alleles in the coding region of resistance genes in 155 rice materials, develops molecular markers at the sites with the strongest specificity; through multiple positive and negative controls or resequencing tests of partial results, develops effective molecular markers for eight resistance genes, namely, Pit, Pish, Pib, Pid3, Pi5, Pia, Pi54 and Pita2 / Ptr, and clarifies the composition of these resistance genes in 109 commonly used breeding parents in the Sichuan Basin. Among them, the Pia gene does not exist in these parents, and the Pit, Pish, Pi54, Pid3, Ptr / Pita2, Pi5 and Pib genes exist in 3.67%, 13.76%, 14.68%, 18.35%, 24.77%, 26.61% and 38.53% of the parents, respectively. In addition, 21.10% of the rice parents did not contain these genes, 35.78% of the parents contained only one resistance gene, and 43.12% of the parents aggregated 2-4 resistance genes.

[0040] The specific beneficial effects include the following:

[0041] 1. Economic benefits of resistance breeding:

[0042] Resistance breeding can significantly reduce the use of chemical pesticides, lower pesticide input costs, and reduce environmental pollution, which is in line with the requirements of sustainable agricultural development.

[0043] Resistant varieties can reduce crop yield losses and improve farmers' economic benefits. Rice blast is one of the main diseases of rice, causing annual yield losses of up to 10%-35%. Breeding rice blast-resistant varieties can significantly reduce related losses.

[0044] 2. Identification and combination of resistance genes:

[0045] The molecular marker primers provided by this technical solution can accurately identify the composition of resistance genes in rice materials. Through PCR amplification and molecular marker technology, it is possible to quickly detect whether a specific gene exists in the material. This is of key significance for identifying the genetic background of resistance genes, analyzing their genetic diversity, and determining the importance of related gene combinations.

[0046] In resistance breeding, the aggregation of multiple resistance genes often provides stronger disease resistance. By clarifying the combination of resistance genes in parental materials, parents can be better selected for hybridization and the resistance of new varieties can be improved.

[0047] 3. Advancement of molecular assisted breeding:

[0048] Molecular marker-assisted selection is a method of using molecular marker technology to quickly and accurately screen and identify target genes. The molecular marker primers provided by this technical solution can be used to identify resistance genes, providing an accurate and reliable molecular marker tool for molecular assisted breeding.

[0049] Molecular assisted breeding can accelerate the process of breeding new varieties, save breeding cycles and costs, and improve breeding efficiency. Especially in the breeding of resistance gene combinations, the application of molecular markers can accurately identify the genetic status of genes and improve the success rate of gene combinations.

[0050] 4. Study on the genetic background of resistance genes:

[0051] Through the application of molecular markers, the genetic background and genetic laws of resistance genes can be further studied. Analyzing gene polymorphism and genetic diversity will help to deeply understand the function and mechanism of resistance genes.

[0052] Research on the genetic background of resistance genes helps to predict and evaluate the stability and persistence of resistance and provide theoretical guidance for resistance breeding.

[0053] 5. Layout and prevention and control strategies of resistant varieties:

[0054] By clarifying the combination of resistance genes in different varieties, we can provide a scientific basis for green prevention and control of rice disease resistance and variety layout. According to the toxicity characteristics of rice blast fungi in different regions, selecting suitable resistance gene combinations can better prevent and control the occurrence and spread of rice blast.

[0055] Accurately understanding the disease resistance of different resistance genes or gene combinations in different regions will help to develop targeted disease prevention and control strategies and provide farmers with scientific planting recommendations.

[0056] In summary, this technical solution provides important professional support for rice resistance breeding through the development and application of molecular marker primers. It can accelerate the identification and combination of resistance genes, promote the application of molecular assisted breeding, further study the genetic background of resistance genes, and provide a scientific basis for the layout of resistance varieties and prevention and control strategies. These effects will have a positive impact on rice disease resistance breeding and the sustainable development of the rice industry.

[0057] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0059] Figure 1 Schematic diagram of the location and application of Pit, Pish and Pib gene molecular markers. A. Pit gene structure and the location of molecular markers. LTR is the long terminal repeat retrotransposon. B. Partial results of PCR detection of rice materials by molecular marker primer pair Pit-pF and Pit-pR. M is the DNA molecular weight standard. C and D. Pish (C) and Pib (D) gene structure and the location of molecular markers. Rectangles are exons. The solid line before the start codon represents the promoter. The introns are between the exons. The 3' non-coding region is after the terminator. E. PCR amplification results of the three exons of the Pib gene in Mianhui 365;

[0060] Figure 2 Schematic diagram of the location of molecular markers of Pid3, Pi5-1 and Pia (RGA5) genes, A-C. The structure of Pid3 (A), Pi5-1 (B) and Pia (C) genes and the location of molecular markers;

[0061] Figure 3 The schematic diagram of the location and application of Pi54 and Pita2 / Ptr gene molecular markers, A. The structure of Pi54 gene and the location of molecular markers. B. The results of partial PCR amplification of the Pi54-mF and Pi54-mR2 molecular marker primers in the rice materials tested, M is the DNA molecular weight standard. C. The structure of Pita2 / Ptr gene and the location of molecular markers;

[0062] Figure 4 The distribution diagram of eight resistance genes in parents in Sichuan Basin. A. The proportion of resistance genes in rice parents. B. The proportion of resistance genes in rice parents. DETAILED DESCRIPTION

[0063] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] Example 1

[0065] The rice blast resistance gene specific molecular marker primer set described in this embodiment comprises:

[0066] The forward primer is Pit-pF, and the reverse primer is Pit-pR, and their nucleotide sequences are shown in SEQ ID NO.1-2.

[0067] The forward primer is Pit-mF, and the reverse primer is Pit-mR, and their nucleotide sequences are shown in SEQ ID NO.3-4.

[0068] The forward primer is Pish / Pi37-F, and the reverse primer is Pish / Pi35-R, and their nucleotide sequences are shown in SEQ ID NO.5-6.

[0069] The forward primer is Pib-F2, and the reverse primer is Pib-R2, and their nucleotide sequences are shown in SEQ ID NO.7-8.

[0070] The forward primer is Pib-mF, and the reverse primer is Pib-mR, and their nucleotide sequences are shown in SEQ ID NO.9-10.

[0071] The forward primer is Pid3-F2, and the reverse primer is Pid3-R2, and their nucleotide sequences are shown in SEQ ID NO.11-12.

[0072] The forward primer is Pid3-mF, ​​and the reverse primer is Pid3-mR, and their nucleotide sequences are shown in SEQ ID NO.13-14.

[0073] The forward primer is Pi5-1-gF, and the reverse primer is Pi5-1-gR, and their nucleotide sequences are shown in SEQ ID NO.15-16.

[0074] The forward primer is Pi5-1-2F, and the reverse primer is Pi5-1-2R, and their nucleotide sequences are shown in SEQ ID NO.17-18.

[0075] The forward primer is Pia(RGA5)-F, and the reverse primer is Pia(RGA5)-R, and their nucleotide sequences are shown in SEQ ID NO.19-20.

[0076] The forward primer is Pi54-F, and the reverse primer is Pi54-R, and their nucleotide sequences are shown in SEQ ID NO.21-22.

[0077] The forward primer is Pi54-mF, and the reverse primer is Pi54-mR2, and their nucleotide sequences are shown in SEQ ID NO.23-24.

[0078] The forward primer is Ptr / Pi-ta2-F, and the reverse primer is Ptr / Pi-ta2-R, and their nucleotide sequences are shown in SEQ ID NO.25-26.

[0079] The forward primer is Ptr / Pi-ta2-F, and the reverse primer is Ptr-Rs, and their nucleotide sequences are shown in SEQ ID NOs.25 and 27.

[0080] On the other hand, the present invention provides a method for detecting rice blast resistance alleles based on the above primers, comprising the following steps:

[0081] S1: Extract genomic DNA of rice samples to be tested;

[0082] S2: performing PCR amplification using the primer pair described in claim 1;

[0083] S3: Detection of Pit gene based on primer pair Pit-pF, Pit-pR and primer pair Pit-mF, Pit-mR;

[0084] S4: Detection of the Pish gene based on primer pairs Pish / Pi37-F and Pish / Pi35-R;

[0085] S5: Detection of Pib gene based on primer pair Pib-F2, Pib-R2 and primer pair Pib-mF, Pib-mR;

[0086] S6: Detection of Pid3 gene based on primer pair Pid3-F2, Pid3-R2 and primer pair Pid3-mF, ​​Pid3-mR;

[0087] S7: Detection of Pi5 gene based on primer pair Pi5-1-gF, Pi5-1-gR and primer pair Pi5-1-2F, Pi5-1-2R;

[0088] S8: Detection of Pia gene based on primer pairs Pia(RGA5)-F and Pia(RGA5)-R;

[0089] S9: Detection of Pi54 gene based on primer pair Pi54-F, Pi54-R and primer pair Pi54-mF, Pi54-mR2;

[0090] S10: Detection of Ptr / Pi-ta2 gene based on primer pairs Ptr / Pi-ta2-F, Ptr / Pi-ta2-R and primer pairs Ptr / Pi-ta2-F, Ptr-Rs.

[0091] In this embodiment, the rice DNA extraction in step S1 specifically includes: 100 mg of rice leaves are placed in a 1.5 mL EP tube, liquid nitrogen is added to grind, 500 μl of CTAB solution is added, and the mixture is placed in a 65°C oven for 30 minutes, and mixed every 10 minutes. After cooling to room temperature, 500 μl of chloroform / isoamyl alcohol (24:1) solution is added, vortexed and mixed, and then allowed to stand at room temperature for 5 minutes. Then centrifuge (12000 r / min) for 15 minutes; take the supernatant into a new 1.5 mL EP tube, add an equal volume of anhydrous ethanol, mix, and stand in a -20°C refrigerator for 10 minutes. Centrifuge (12000 r / min) for 10 minutes, discard the supernatant, wash the precipitate twice with 1000 ml of pre-cooled 75% anhydrous ethanol, add 100 μl of ddH2O to dissolve the DNA after the precipitate is dried, and store in a -20°C refrigerator for use.

[0092] In this embodiment, the PCR reaction system for PCR amplification in step S2 is: 2.0 μL template DNA (400 ng / μL), 2.0 μL Buffer, 0.5 μL dNTP (2.5 mM each), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 0.5 μL Taq enzyme (2.5 U / μL), 14.0 μL ddH2O, a total of 20 μL.

[0093] The invention also discloses the application of the method in rice material gene typing and rice blast resistance breeding.

[0094] The invention also discloses a kit containing the primer pair.

[0095] The invention also discloses the application of the kit in rice material gene typing and rice blast resistance breeding.

[0096] Example 2

[0097] Rice materials for testing

[0098] The present invention uses 136 rice materials, which are composed of 24 rice blast resistance single gene lines, 109 commonly used breeding parents in the Sichuan Basin (25-133), and 3 control materials: Nipponbare (NPB), Zhonghua11 (ZH11) and Lijiangxintuanheigu (LTH) (Table 1).

[0099] Table 1 Tested rice varieties

[0100]

[0101]

[0102]

[0103] Screening of nucleotide polymorphism sequences and primer design

[0104] The CDS sequences of eight target genes, Pit (AB379816.1), Pish (XM_015790911), Pib (AB013449.2), Pid3 (FJ745364.1), Pi5-1 (EU869185.1), Pia (AB604627.1), Pi54 (AY914077) and Pita2 / Ptr (MG385185), were downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The sequence differences of the target genes and the alleles in the genomes of 155 rice resources (including indica rice, japonica rice, African cultivated rice and wild rice, etc.) in the database of the Rice Resource Center (http: / / ricerc.sicau.edu.cn / ) were then compared to discover specific polymorphic sites and develop molecular markers at the most specific positions. Among them, when a specific single nucleotide is used as the 3' end of the molecular marker primer, the specificity of PCR amplification is relatively poor, and non-specific amplification often occurs. Therefore, when designing such primers, the second base adjacent to the SNP is generally specifically mutated to form an additional mismatch site, which can significantly improve the specificity of PCR amplification.

[0105] Rice DNA extraction

[0106] Extract rice DNA using the CTAB method: Take 100 mg of rice leaves, put them in a 1.5 mL centrifuge tube, and then add liquid nitrogen to grind. Add 500 μL of CTAB solution and mix well, place in a 65°C oven for 30 minutes, and mix every 10 minutes. Transfer to room temperature to cool, then add 500 μL of chloroform / isoamyl alcohol (24:1) solution, mix well and let stand for 5 minutes. Then put it in a centrifuge, centrifuge at 12000r / min for 15 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, and add an equal volume of anhydrous ethanol to precipitate DNA. After mixing, put it in a centrifuge, centrifuge at 12000r / min for 10 minutes, remove the supernatant, wash the precipitate twice with 75% anhydrous ethanol, and add 100 μL of ddH2O to dissolve the DNA precipitate after drying for 30 minutes. After mixing, put it in a -20°C refrigerator for later use.

[0107] PCR amplification conditions and accuracy test of molecular markers

[0108] The DNA polymerase used for PCR amplification was from Beijing Zhuangmeng International Biogene Technology Co., Ltd. (Cat. No.: ZT101). The PCR amplification system included 2.0 μL template DNA (400 ng / μL), 2.0 μL Buffer, 0.5 μL dNTP (2.5 mM each), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 0.5 μL Taq enzyme (2.5 U / μL), and 14.0 μL ddH2O, for a total of 20 μL.

[0109] In order to improve the accuracy of molecular markers, positive control materials containing each target gene were first screened in rice blast resistant single gene lines and rice with known full gene sequences (Shuhui 498, Shuhui 527, Shuhui 548, Digu B, Yahui 2115, Yixiang 1B, Huazhan, Wushan Simiao, Zhonghua 11, Nihonbare and LTH). For example, the single gene line IRBLb-B contains the resistance gene Pib, NPB contains the Pish gene, and LTH, as a susceptible material, does not contain the resistance gene. The PCR amplification conditions were adjusted to the optimal level (Table 2). In the final identification results, some materials were randomly selected for target gene sequencing to verify the accuracy of the molecular markers.

[0110] Table 2 Molecular marker primer pairs and PCR amplification conditions

[0111]

[0112]

[0113]

[0114] Example 3

[0115] Results and Analysis

[0116] Development and application of molecular markers for Pit, Pish and Pib genes

[0117] A long terminal repeat (LTR) retrotransposon was inserted into the promoter of the rice resistance gene Pit, which significantly increased the expression of the Pit gene, thereby conferring resistance to rice blast in the rice material K59

[12] . Based on this characteristic, the forward primer Pit-pF of the Pit gene molecular marker was designed in the LTR, and the reverse primer Pit-pR was located in the promoter region adjacent to the start codon ( Figure 1 A). Using this primer pair, the Pit gene was only detected in 4 of the tested materials ( Figure 1 B). In order to improve the accuracy of the test, another pair of primers, Pit-mF and Pit-mF, were developed based on specific SNP sites in the gene coding region. The test results were consistent with the Pit-pF and Pit-pF primer pairs. The Pit gene was only detected in four rice materials tested, namely E33, Hanghui 1179, Yuenong Si Miao and Thai Xiangdao, indicating that both pairs of primers have strong specificity. Yuenong Si Miao was randomly selected from the four positive materials to amplify the full length of the Pit gene and sequence it, and it was found that its DNA sequence was consistent with the Pit gene.

[0118] The specific primer pair for Pish gene molecular marker is located at the end of the gene ( Figure 1 C), 16 materials were found to contain the Pish gene, including Nipponbare (NPB) and the single gene line IRBLsh-B. The Pish gene was originally cloned from Nipponbare

[13] , and IRBLsh-B is a single gene line containing the Pish gene that is resistant to rice blast

[14] . In addition, there were 14 breeding parent materials commonly used in the Sichuan Basin (Table 3).

[0119] The specific primer pair for Pib gene molecular marker is located in the third exon of the gene ( Figure 1 D), in order to enhance the specificity of molecular markers, two pairs of specific primers were developed at four specific sites to jointly mark the Pib gene. It was found that 43 materials could simultaneously amplify these two molecular markers, indicating that they contained the Pib gene (Table 4), including the positive rice blast resistance single gene line IRBLb-B (Pib gene), and the other 42 were breeding parents, among which the genomes of Huazhan, Wushan Simiao, Yahui 2115 and Yixiang 1B had been sequenced. Through sequence comparison, it was found that they all contained the Pib gene, which was consistent with the marker identification results. In addition, the parent material Mianhui 365 was randomly selected, and the exons of its Pib gene were amplified and sequenced ( Figure 1E), it was clear that its DNA sequence was consistent with the Pib gene, indicating that the molecular marker had good specificity.

[0120] Table 3 Rice materials containing Pish gene

[0121]

[0122] Table 4 Rice materials containing Pib gene

[0123]

[0124]

[0125] Development and application of molecular markers for Pid3, Pi5-1 and Pia genes

[0126] The two molecular marker primer pairs of Pid3 gene are located at the end of the gene coding region and jointly identify Pid3 gene ( Figure 2 A). After testing, it was found that 20 rice materials tested could simultaneously amplify two markers, indicating that they contained the Pid3 gene (Table 5), including Huazhan, Shuhui 548, Wushan Simiao and Yahui 2115. Analysis of the genome sequences of these four materials revealed that they all contained the Pid3 gene.

[0127] The Pi5 gene locus is composed of two NBS-LRR genes, Pi5-1 and Pi5-2, which work together to confer broad-spectrum resistance to rice blast. Among them, the promoter of the Pi5-1 gene varies greatly among different alleles, so two pairs of molecular marker primers were developed at four specific nucleotide sites in its promoter and coding region to mark the Pi5 gene locus ( Figure 2 B). After testing, it was found that 30 rice materials tested could be labeled by these two pairs of primers (Table 6), indicating that they contained the Pi5-1 gene, including the positive disease resistance single gene line IRBL5-M (Pi5 gene) and 29 breeding parents. Among these parents, Huazhan, Shuhui 498 and Shuhui 548 had full genome sequences, and analysis showed that they all contained the Pi5 gene pair.

[0128] The Pia gene locus is also composed of two "head-to-head" NBS-LRR genes RGA4 and RGA5. The RGA4 gene is a "helper" with a relatively conservative sequence, while the resistance protein encoded by the RGA5 gene has the function of sensing effector factors and has a relatively abundant sequence polymorphism, which is mainly concentrated in the HMA (heavy metal-associated domain) domain at the C-terminus. Therefore, the molecular marker primer pair for identifying the Pia gene was designed in the HMA domain of the RGA5 gene ( Figure 2C). After testing, only the single gene line IRBLa-A (Pia gene) was able to amplify the molecular marker, indicating that other test materials did not contain the Pia gene.

[0129] Table 5 Rice materials containing Pid3 gene

[0130]

[0131]

[0132] Table 6 Rice materials containing Pi5-1 gene

[0133]

[0134]

[0135] Development and application of Pi54 and Pita2 / Ptr gene molecular markers

[0136] Based on the specific nucleotide sites, two molecular marker primer pairs were designed in the coding region of Pi54 gene to jointly identify the Pi54 gene contained in the test materials ( Figure 3 A). After testing, it was found that 16 rice parents contained the Pi54 gene ( Figure 3 B and Table 7), which included Yixiang 1B, whose genome contained the Pi54 gene, indicating that the molecular marker can effectively detect the Pi54 gene.

[0137] The Pita2 and Ptr genes were cloned in IR64 and Katy materials, respectively, and are alleles of each other with the same gene sequence. Among the 28 resistant and susceptible rice materials, the main difference between the Pita2 / Ptr alleles is that there is a 12bp fragment inserted at the 3' end of the susceptible Pita2 / Ptr gene, while the other regions are very conservative. Therefore, the primer pair Pit-F and Pit-R were designed in the conservative regions at both ends of the 12bp difference site to identify the resistant and susceptible Pita2 / Ptr genes. Then, the reverse primer Ptr-Rs was designed with the 12bp insertion sequence of the susceptible type as the core, and paired with Pit-F to amplify the marker that can identify the susceptible Pita2 / Ptr gene. The material that cannot be amplified by PCR contains the Pita2 / Ptr gene ( Figure 3C). It was identified that the primer pair Pit-F and Pit-R could amplify bands from all the materials tested, while the primer pair Pit-F and Ptr-Rs could not amplify bands from 28 materials (Table 8), including the single gene line IRBLta2-Pi containing the Pita2 / Ptr gene, indicating that these 28 materials contain the Pita2 / Ptr gene. In addition, among the parental materials with known genome sequences tested, four materials, Huazhan, Shuhui 548, Shuhui 527 and Yixiang 1B, contain the Pita2 / Ptr gene, which is consistent with the results of molecular marker identification, indicating that the molecular marker of the Pita2 / Ptr gene has good accuracy.

[0138] Table 7 Rice materials containing Pi54 gene

[0139]

[0140] Table 8 Rice materials containing Pita2 / Ptr gene

[0141]

[0142]

[0143] Distribution of eight resistance genes in parents in Sichuan Basin

[0144] According to the results of molecular marker identification, it was found that the Pia gene could not be detected in the parents in the Sichuan Basin, while the presence ratios of the other seven resistance genes Pit, Pish, Pi54, Pid3, Ptr / Pita2, Pi5 and Pib in these parents were 3.67%, 13.76%, 14.68%, 18.35%, 24.77%, 26.61% and 38.53%, respectively. Figure 4 A), in addition, 21.10% of rice parents do not contain these genes ( Figure 4 B). 35.78% of the parents contained only one resistance gene. In addition, some parents aggregated 2, 3 and 4 resistance genes, accounting for 27.52%, 13.76% and 1.83% of the tested parents respectively. Among them, Huazhan (Pib, Pi5, Ptr / Pita2 and Pid3) and Hanghui 1179 (Pit, Pi5, Ptr / Pita2 and Pid3) aggregated 4 resistance genes (Table 3-8).

[0145] In order to enable molecular markers to break through the limitations of rice genetic background, accurately identify resistance genes, and thus clarify the resistance gene composition in parents, the present invention sets multiple positive and negative controls in the process of testing molecular markers. Using the general variety LTH, and other varieties of whole genome sequencing among the test varieties, Shuhui 498, Shuhui 527, Shuhui 548, Digu B, Yahui 2115, Yixiang 1B, Huazhan, Wushan Simiao, Zhonghua 11 and Nihonbare, their gene sequences are analyzed to clarify the positive and negative control materials of each resistance gene, effectively ensuring the accuracy of molecular markers. When a molecular marker cannot completely distinguish between positive and negative controls, more molecular markers will be introduced for joint identification. Therefore, except for Pish and Pia genes, the other six genes all require two molecular markers for joint identification. In addition, when designing molecular marker identification primers at single nucleotide difference sites, the second base at the 3' end will be mutated, specific mismatched bases will be introduced, and the specificity of PCR amplification will be enhanced. Similar mutation methods have good applications in the development of PigmR gene molecular markers.

[0146] In addition to the genes at the Pi2 and Pik loci, the Pia, Pit, Pish, Pi54, Pid3, Ptr / Pita2, Pi5 and Pib genes are widely distributed in various rice-growing areas across the country, and show different distribution characteristics in different areas. For example, the Pib gene is most widely distributed in Liaoning, Shanghai and Huanghuaihai rice-growing areas, while the Pi5 gene is most widely distributed in Chongqing rice-growing areas. The present invention found that the Pib gene is most widely distributed in rice varieties in the Sichuan Basin rice-growing area ( Figure 4A), while the Pia gene was not detected in the rice parents tested, indicating that the utilization rate of the Pia gene in rice resistance breeding in the Sichuan Basin is very low. This is negatively correlated with the presence rate of their corresponding avirulent genes AvrPib and AvrPia in the rice blast fungus population in the Sichuan Basin. The AvrPib gene exists in the vast majority of rice blast fungi, while the Avr-Pia gene only exists in a small number of rice blast fungi. Therefore, the Pib gene can play a resistance function and is used by most rice parents, while the Pia gene is difficult to play a disease resistance function and is abandoned. In addition, these resistance genes still have good rice blast resistance functions in different rice areas. For example, in the rice blast resistance identification base for new varieties in the rice regional trials in Taohu, Hunan, Hunan Province and the middle and lower reaches of the Yangtze River, the disease-resistant single gene lines containing Ptr / Pita2 and the single gene lines containing Piz5 and Pikm genes showed good field resistance to seedling leaf blast and panicle neck blast. In addition, gene aggregation can exert a stronger disease resistance function. Therefore, through molecular marker identification, the composition of resistance genes in parents in the Sichuan Basin can be revealed, the application of parents in resistance breeding can be promoted, and the efficiency of resistance gene introgression and aggregation can be improved through molecular assisted breeding. In addition, the composition of resistance genes can be clarified according to the disease conditions of various varieties in rice production, which can reveal the disease resistance of each resistance gene or resistance gene combination in different regions, and provide guidance for green prevention and control of rice disease resistance, variety layout and disease resistance breeding.

[0147] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A rice blast resistance gene specific molecular marker primer set, characterized in that the primer set include: Forward primer Pit-pF, reverse primer Pit-pR, whose nucleotide sequences are shown in SEQ ID NO.1-2; Forward primer Pit-mF, reverse primer Pit-mR, the nucleotide sequences of which are shown in SEQ ID NO.3-4; Forward primer Pish / Pi37-F, reverse primer Pish / Pi35-R, the nucleotide sequences of which are shown in SEQ ID NO.5-6; Forward primer Pib-F2, reverse primer Pib-R2, the nucleotide sequences of which are shown in SEQ ID NO.7-8; Forward primer Pib-mF, reverse primer Pib-mR, the nucleotide sequences of which are shown in SEQ ID NO.9-10; Forward primer Pid3-F2, reverse primer Pid3-R2, whose nucleotide sequences are shown in SEQ ID NO.11-12; Forward primer Pid3-mF, ​​reverse primer Pid3-mR, the nucleotide sequences of which are shown in SEQ ID NO.13-14; Forward primer Pi5-1-gF, reverse primer Pi5-1-gR, the nucleotide sequences of which are shown in SEQ ID NO.15-16; Forward primer Pi5-1-2F, reverse primer Pi5-1-2R, the nucleotide sequences of which are shown in SEQ ID NO.17-18; Forward primer Pia(RGA5)-F, reverse primer Pia(RGA5)-R, whose nucleotide sequences are shown in SEQ ID NO.19-20; Forward primer Pi54-F, reverse primer Pi54-R, whose nucleotide sequences are shown in SEQ ID NO.21-22; Forward primer Pi54-mF, reverse primer Pi54-mR2, the nucleotide sequences of which are shown in SEQ ID NO.23-24; Forward primer Ptr / Pi-ta2-F, reverse primer Ptr / Pi-ta2-R, whose nucleotide sequences are shown in SEQ ID NO.25-26; and The forward primer is Ptr / Pi-ta2-F, and the reverse primer is Ptr-Rs, and their nucleotide sequences are shown in SEQ ID NOs.25 and 27.

2. A method for detecting rice blast resistance alleles based on the primer set of claim 1, characterized in that: The following steps are involved: S1: Extract genomic DNA of rice samples to be tested; S2: performing PCR amplification using the primer set described in claim 1; S3: Detection of Pit gene based on primer pair Pit-pF, Pit-pR and primer pair Pit-mF, Pit-mR; S4: Detection of the Pish gene based on primer pairs Pish / Pi37-F and Pish / Pi35-R; S5: Detection of Pib gene based on primer pair Pib-F2, Pib-R2 and primer pair Pib-mF, Pib-mR; S6: Detection of Pid3 gene based on primer pair Pid3-F2, Pid3-R2 and primer pair Pid3-mF, ​​Pid3-mR; S7: Detection of Pi5 gene based on primer pair Pi5-1-gF, Pi5-1-gR and primer pair Pi5-1-2F, Pi5-1-2R; S8: Detection of Pia gene based on primer pairs Pia(RGA5)-F and Pia(RGA5)-R; S9: Detection of Pi54 gene based on primer pair Pi54-F, Pi54-R and primer pair Pi54-mF, Pi54-mR2; S10: Detection of Ptr / Pi-ta2 gene based on primer pairs Ptr / Pi-ta2-F, Ptr / Pi-ta2-R and primer pairs Ptr / Pi-ta2-F, Ptr-Rs.

3. Application of the detection method as claimed in claim 2 in rice material genotyping and rice blast resistance breeding.

4. A kit comprising the primer set according to claim 1.

5. Use of the kit as claimed in claim 4 in rice material genotyping and rice blast resistance breeding.

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