Rice resistance to root-knot nematode and its molecular marker

Abstract

The application discloses a main QTL of rice paratrichodorus reniformis resistance and a molecular marker and application, and belongs to the technical field of rice breeding and molecular biology. The application takes the paratrichodorus reniformis resistant variety 685 and the disease susceptible variety Minghui 63 as parents to construct an F2 genetic population, determines the main QTL of the rice paratrichodorus reniformis resistance by using the population through the BSA method, simultaneously finds that two SNP sites exist in the main QTL locating interval, constructs a KASP molecular marker according to the above sites, and the KASP molecular marker can be successfully applied to the verification of the rice paratrichodorus reniformis resistance. The application can be used for molecular assisted breeding of rice, accelerates the rice breeding process, is simple, safe and effective, is beneficial to improving the economic value of the rice variety, and is suitable for large-scale popularization and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rice breeding and molecular biology, and more particularly to a major QTL and molecular marker for rice resistance to Pratylenchus spp. and application thereof. BACKGROUND

[0002] Pratylenchus spp. Meloidogyne graminicola is one of the most serious plant nematode pathogens that harm rice production, with extremely strong ecological adaptability. In particular, with the popularization of dry direct seeding technology and the promotion of water-saving cultivation system, the microecological environment of the rice field has been significantly changed, creating favorable conditions for the spread of nematode populations.

[0003] The current prevention and control measures for Pratylenchus spp. have the limitations of unstable prevention and control effect and high operation cost in field practice. Under this background, breeding of resistant varieties has become the most cost-effective control strategy. Traditional breeding strategies for resistant varieties mainly rely on phenotype-oriented screening, but this method has inherent defects such as long screening period, significant environmental interference, large phenotype evaluation bias, and low selection efficiency.

[0004] Molecular marker-assisted breeding technology realizes targeted selection through accurate genotyping, not only significantly reducing breeding costs and shortening the breeding period of disease-resistant varieties, but also achieving multiple resistance gene aggregation, thereby improving genetic gain and producing significant social and economic value. With the rapid development of high-throughput sequencing technology, molecular marker systems based on single nucleotide polymorphism (SNP) between individuals have become a core tool for crop genetic research. Among them, the KASP (Kompetitive Allele Specific PCR) marker technology, with its unique design principle, uses fluorescently labeled probes to specifically amplify target SNP sites, and genotypes by scanning fluorescent signals. This technology breaks through the technical bottleneck of traditional detection, has the advantages of high-throughput, high-precision, low-cost, and full-process automation, and has played a key role in fine mapping and molecular design breeding of important crops such as rice. However, there is little research on the screening of major QTLs for Pratylenchus spp. resistance in rice.

[0005] Therefore, how to fine map the major QTL for Pratylenchus spp. resistance in rice and develop KASP molecular markers linked thereto has become a problem that needs to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a major QTL and molecular marker for Pratylenchus spp. resistance in rice and application thereof.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] The first object of the present application is to provide a major QTL for rice paratrichodorus reniformis resistance, which is located on chromosome 11 of rice and named as qMg11-1, and the positioning interval is 29.03-33.82 Mb.

[0009] Still another object of the present application is to provide KASP molecular markers for the major QTL for rice paratrichodorus reniformis resistance, including closely linked molecular markers 2902A and 3382B; wherein,

[0010] The primer set sequence for detecting the 2902A is as follows:

[0011] 2902A-F1: 5'-GAAGGTGACCAAGTTCATGCTAAGGAACACTGAATG

[0012] TTCAAAACAT-3', SEQ ID NO. 1;

[0013] 2902A-F2: 5'-GAAGGTCGGAGTCAACGGATTAAGGAACACTGAAT

[0014] GTTCAAAACAC-3', SEQ ID NO. 2;

[0015] 2902A-R: 5'-TTAGCTAAACAGTAAGTGTAAGTTAAAAT-3', SEQ ID NO. 3;

[0016] The primer set sequence for detecting the 3382B is as follows:

[0017] 3382B-F1: 5'-GAAGGTGACCAAGTTCATGCTGGTATCTCACTATA

[0018] AATACCCAAAG-3', SEQ ID NO. 4;

[0019] 3382B-F2: 5'-GAAGGTCGGAGTCAACGGATTGGTATCTCACTATAA

[0020] ATACCCAAAC-3', SEQ ID NO. 5;

[0021] 3382B-R: 5'-AATCGAGTAGCTAGCTTTTTGCTTAAACC-3', SEQ ID NO. 6.

[0022] Another objective of this application is to provide the application of the above-mentioned major QTLs of resistance to rice root-knot nematodes in the breeding of rice varieties resistant to root-knot nematodes of the Poaceae family.

[0023] Another object of this application is to provide: the application of the above-mentioned KASP molecular marker, wherein the application is in any of the following directions:

[0024] 1) Application in the breeding of rice varieties resistant to root-knot nematodes of the Poaceae family;

[0025] 2) Application in detecting resistance to root-knot nematodes in rice (Poaceae family);

[0026] 3) Application in detecting the major QTL qMg11-1 for resistance to root-knot nematodes in rice (Poaceae family);

[0027] 4) Application in the preparation of a kit for detecting resistance to root-knot nematodes in rice.

[0028] Another object of this application is to provide a kit for detecting resistance to root-knot nematodes in rice (Poaceae family), comprising the aforementioned KASP molecular marker.

[0029] Another objective of this application is to provide a method for detecting resistance to root-knot nematodes in rice, comprising: extracting rice DNA, using the aforementioned KASP molecular marker to amplify the DNA by PCR, detecting the amplification product by fluorescence quantitative PCR, and analyzing the resistance to root-knot nematodes in rice based on the detection results.

[0030] As a preferred technical solution, the PCR amplification reaction system is 10 μL, as detailed below:

[0031] 5 μL of FLU-ARMS 2× PCR Mix, 2 μL of genomic DNA, 0.75 μL of Primer mix, and 2.25 μL of ddH2O; the concentration ratio of primers F1, F2, and R in the Primer mix was 1:1:3.

[0032] As a preferred technical solution, the PCR amplification reaction procedure is as follows:

[0033] Pre-denaturation at 95 ℃ for 5 min; amplification at 95 ℃ for 5 s, 58 ℃ for 20 s for 40 cycles; plate reading at 30 ℃ for 30 s for 1 cycle.

[0034] As a preferred technical solution, the following principles govern the interpretation of detection results after KASP molecular marker amplification:

[0035] If the test result of 2902A is blue or the test result of 3382B is blue, it is determined that the rice does not contain the root-knot nematode resistance site qMg11-1.

[0036] If the test result of 2902A is red or the test result of 3382B is red, it is determined that the rice contains the root-knot nematode resistance site qMg11-1.

[0037] If the test result of 2902A is green or the test result of 3382B is green, the material to be tested contains the heterozygous root-knot nematode resistance site qMg11-1.

[0038] Another object of this application is to provide the application of the above-described reagent kit or method in the detection of resistance to root-knot nematodes in rice (Poaceae family).

[0039] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0040] This invention constructs an F2 genetic population using the resistant rice variety 685 (resistant to root-knot nematodes of the Poaceae family) and the susceptible variety Minghui 63 (MH63) as parents. Using this population, major QTLs for resistance to root-knot nematodes in rice were determined through BSA and fine mapping. Two SNP loci were found within the mapping interval of these major QTLs. Based on these loci, a KASP molecular marker was constructed and successfully applied to verify rice resistance to root-knot nematodes of the Poaceae family. This invention accelerates the rice breeding process, is simple and easy to implement, safe and effective, and is beneficial to improving the economic value of rice varieties, making it suitable for large-scale promotion and application. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0042] Figure 1 For: Location information of major QTLs of grass root-knot nematodes.

[0043] Figure 2 The results show the quantitative association between the KASP molecular marker 2902A and the phenotype; blue represents homozygous susceptibility gene, red represents homozygous resistance gene, and green represents heterozygous phenotype; A1 is the susceptible parent Minghui 63, A2-B3 are susceptible single plants; F1 is the resistant parent 685, F2-H12 are resistant single plants; B4-E12 are heterozygous single plants.

[0044] Figure 3The results show the quantitative association between the KASP molecular marker 3382B and the phenotype; blue represents homozygous susceptibility gene, red represents homozygous resistance gene, and green represents heterozygous phenotype; A1 is the susceptible parent Minghui 63, A2-B3 are susceptible single plants; F2 is the resistant parent 685, F2-H12 are resistant single plants; B4-F1 are heterozygous single plants. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0047] Rice 685 is "Transcriptome and co-expression network a'analyses ofresistant and susceptible rice cultivars in response to" in MDPI Meloidogyne graminicola The rice materials disclosed in the patent application are currently held by the Rice Research Institute of the Guangdong Academy of Agricultural Sciences and can be made available to the public in order to meet the requirements of sufficient patent disclosure.

[0048] Minghui 63 (MH63) is a conventional rice variety with wide applications. Almost every laboratory has its own records. The rice material is also disclosed in the article "A 9.5-kb deletion in the 1st intron of OsMADS51 enhances temperature sensitivity in rice". It is currently held by the Rice Research Institute of Guangdong Academy of Agricultural Sciences and can be made available to the public to meet the requirements of sufficient patent disclosure.

[0049] Example 1

[0050] Localization of the resistance site qMg11-1 in the root-knot nematode of the Poaceae family

[0051] 1. Acquisition of experimental materials

[0052] In the screening of rice germplasm materials for resistance to root-knot nematodes of the Poaceae family, the resistant material 685 was finally identified as a resistant variety. 685 was then crossed with the susceptible variety Minghui 63 (MH63) to obtain F1. The genotype of the F1 rice was used to verify whether the hybridization was successful. The F1 rice was self-pollinated to obtain the F2 genetic population, and the F2:3 family was harvested to verify the resistance phenotype.

[0053] 2. BSA yielded the resistance locus qMg11-1 for root-knot nematodes of the Poaceae family.

[0054] To uncover resistance information in root-knot nematodes of the Poaceae family, this application used the BSA method to determine resistance loci, as detailed below:

[0055] (1) F2 (685 / MH63) population resistance classification

[0056] F2 seeds were routinely germinated and transplanted into hydroponic boxes. After one week of seedling growth, they were transplanted into pots. Simultaneously, nematodes were collected and hatched based on the number of rice plants. After the rice plants had adapted to growing in the pots for one week, a nematode suspension was magnetically stirred at low speed, and a measured amount of nematodes was injected into the pots. Phenotypic characteristics could be observed after 15 days. The number of root knots in the rice parents 685 and MH63 was first counted. Based on the average number of root knots in the resistant parents, the F2 generation plants (685 / MH63) were classified as resistant or susceptible.

[0057] The specific criteria for classifying immune system resistance are as follows:

[0058] F2 generation plants inoculated with root-knot nematodes of the Poaceae family, those with a lower number of root knots than the average number of root knots of the parent MH63 were considered to have a resistant phenotype.

[0059] F2 generation plants inoculated with root-knot nematodes of the Poaceae family, those with a root knot number greater than or equal to the average root knot number of the parent MH63 were classified as having an extremely sensitive phenotype.

[0060] (2) BSA method for determining resistance sites

[0061] From the F2 (685 / MH63) population validated in step (1), select 30 plants with extreme resistance (R) and 30 plants with extreme susceptibility (S). Extract high-quality DNA from young rice leaves using the CTAB method and mix them in equal amounts. Perform whole-genome resequencing using the Illumina NovaSeq PE150 platform to obtain effective data: resistance pool ≥30×, susceptibility pool ≥30×, and parents P1 / P2 30× each.

[0062] Analysis using the GATK 4.2 workflow identified 412,759 high-quality SNPs (filtering criteria: QUAL≥30, DP≥10, GQ≥20). ΔSNP-index analysis showed a significant association peak in the 26.18-33.94 Mb region of chromosome 11. Further mapping using exchanged single plants, based on genotype and resistance / susceptibility phenotypes, narrowed the resistance locus region from the 26.18-33.94 Mb region of chromosome 11 in the MH63 reference genome to the 29.03-33.82 Mb region of chromosome 11, defining it as the qMg11-1 locus. Figure 1 The reference genome for MH63 is the sequence MH63RS3.fasta.gz published on the website Rice Information Gateway (http: / / rice.hzau.edu.cn / cgi-bin / rice_rs3 / download_ext).

[0063] Example 2

[0064] Development of KASP markers for the qMg11-1 locus in the root-knot nematode of the Poaceae family

[0065] The study found that within the mapping interval of the major QTL for resistance to root-knot nematodes in rice (gramineous plants), qMg11-1, there are two SNP sites, whose corresponding physical locations are as follows:

[0066] SNP site of KASP molecular marker 2902A: MH63 genome physical diagram 11 chromosome 29028515bp, the base can be T or C, and the amino acid can be His or Pro;

[0067] SNP locus of KASP molecular marker 3382B: MH63 genome physical diagram, chromosome 11, 33827446bp.

[0068] Using the above SNP sites, KASP molecular markers were designed as follows:

[0069] The primer sequence for detecting the KASP molecular marker 2902A is as follows:

[0070] 2902A-F1: 5'-GAAGGTGACCAAGTTCATGCTAAGGAACACTGAATG

[0071] TTCAAAACAT-3', SEQ ID NO.1;

[0072] 2902A-F2: 5'-GAAGGTCGGAGTCAACGGATTAAGGAACACTGAAT

[0073] GTTCAAAACAC-3';SEQ ID NO.2;

[0074] 2902A-R: 5'-TTAGCTAAACAGTAAGTGTAAGTTAAAAT-3', SEQ ID NO.3;

[0075] The primer sequence for detecting the KASP molecular marker 3382B is as follows:

[0076] 3382B-F1:5'-GAAGGTGACCAAGTTCATGCTGGTATCTCACTATA

[0077] AATACCCAAAG-3', SEQ ID NO.4;

[0078] 3382B-F2:5'-GAAGGTCGGAGTCAACGGATTGGTATCTCACTATAA

[0079] ATACCCAAAC-3', SEQ ID NO.5;

[0080] 3382B-R: 5'-AATCGAGTAGCTAGCTTTTTGCTTAAACC-3', SEQ ID NO. 6.

[0081] Example 3

[0082] Molecular marker-assisted detection

[0083] Using the KASP molecular markers designed in Example 2, genomic DNA was extracted from rice leaves and amplified from the resistant parent 685, the resistant pool, the susceptible parent MH63, the susceptible pool, and the F2:3 progeny population.

[0084] The specific method is as follows:

[0085] Preparation of Primer mix: The primers obtained in Example 2 were uniformly diluted to a concentration of 10 μM and prepared into a Primer mix according to the ratio of F1:F2:R=1:1:3.

[0086] The quantitative real-time PCR amplification system (10 μL) consisted of 5 μL of FLU-ARMS 2× PCR Mix, 2 μL of genomic DNA (20 ng / μL), 0.75 μL of Primer mix, and 2.25 μL of ddH2O.

[0087] The quantitative PCR amplification program was as follows: pre-denaturation at 95℃ for 5 min; amplification at 95℃ for 5 s and 58℃ for 20 s for 40 cycles; and reading the plate at 30℃ for 30 s for 1 cycle.

[0088] The real-time PCR results file was exported from the Roche LightCycler 480II real-time PCR instrument. 2902A ( Figure 2 ) and 3382B ( Figure 3 ) Genotype results.

[0089] Results Analysis: Based on Figure 1 and Figure 2 As a result, both 2902A and 3382B were able to distinguish between the resistance and susceptibility types of the root-knot nematode resistance locus qMg11-1. Secondly, the two molecular markers were used to perform genotyping on samples from the F2:3 population (the genotypes of the exchanged F2 plants were identified, and the offspring of these plants were collected for phenotypic identification). The results showed that the resistance and susceptibility genotypes were consistent with the root-knot nematode resistance identification results.

[0090] To more accurately detect the resistance genotype, the genotype was determined by combining the results of 2902A and 3382B, as detailed below:

[0091] (1) If the corresponding base T (blue) is detected in 2902A or blue is detected in 3382B, it is determined that the rice does not contain the root-knot nematode resistance site qMg11-1;

[0092] (2) If the corresponding base C (red) is detected in 2902A or red is detected in 3382B, then it is determined that the rice contains the root-knot nematode resistance site qMg11-1;

[0093] (3) If 2902A detects the bases T and C (green) or 3382B detects green, then the material to be tested contains the heterozygous root-knot nematode resistance site qMg11-1 of the grass family.

[0094] Markers 2902A and 3382B are located on either side of the resistance gene. By simultaneously identifying both markers, the genotype of the resistance gene can be determined more accurately.

[0095] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for detecting KASP molecular markers of resistance to root-knot nematodes in rice (Poaceae family), characterized in that, The kit includes a KASP molecular marker primer set for detecting tightly linked molecular markers 2902A and 3382B. These tightly linked molecular markers 2902A and 3382B are located within the major QTL mapping interval for resistance to root-knot nematodes. The major QTL is named qMg11-1 and is located on chromosome 11 of the rice MH63 reference genome, with a mapping interval of 29.03-33.82 Mb. The primer sequence for detecting the tightly linked molecular marker 2902A is as follows: 2902A-F1:5'-GAAGGTGACCAAGTTCATGCTAAGGAACACTGAA TGTTCAAAACAT-3', SEQ ID NO.1; 2902A-F2: 5'-GAAGGTCGGAGTCAACGGATTAAGGAACACTGAAT GTTCAAAACAC-3', SEQ ID NO.2; 2902A-R: 5'-TTAGCTAAACAGTAAGTGTAAGTTAAAAT-3', SEQ ID NO.3; The primer sequence for detecting the tightly linked molecular marker 3382B is as follows: 3382B-F1:5'-GAAGGTGACCAAGTTCATGCTGGTATCTCACTATA AATACCCAAAG-3', SEQ ID NO.4; 3382B-F2:5'-GAAGGTCGGAGTCAACGGATTGGTATCTCACTATAA ATACCCAAAC-3', SEQ ID NO.5; 3382B-R: 5'-AATCGAGTAGCTAGCTTTTTGCTTAAACC-3', SEQ ID NO.

6.

2. The application of the KASP molecular marker primer set of the tightly linked molecular marker 2902A as described in claim 1, characterized in that, The application is any one of the following: 1) Application in the breeding of rice varieties resistant to root-knot nematodes of the Poaceae family; 2) Application in detecting resistance to root-knot nematodes in rice (Poaceae family); 3) Application in detecting the major QTL qMg11-1 for resistance to root-knot nematodes in rice (Poaceae family); 4) Application in the preparation of a kit for detecting resistance to root-knot nematodes in rice (Poaceae family); When using the KASP molecular marker primer set of the tightly linked molecular marker 2902A, the following criteria are used for judgment: When the 2902A test result is blue, it is determined that the rice does not contain the root-knot nematode resistance site qMg11-1; When the 2902A test result is red, it is determined that the rice contains the root-knot nematode resistance site qMg11-1; When the 2902A test result is green, it is determined that the rice contains the heterozygous root-knot nematode resistance site qMg11-1 of the Poaceae family; The rice variety is either Rice 685 or Minghui 63.

3. A kit for detecting resistance to root-knot nematodes in rice (Poaceae family), characterized in that, The KASP molecular marker primer set includes the tightly linked molecular marker 2902A as described in claim 1.

4. A method for detecting resistance to root-knot nematodes in rice (Poaceae family), characterized in that, The process is as follows: Rice DNA was extracted, and the DNA was amplified by PCR using the KASP molecular marker primer set of the tightly linked molecular marker 2902A as described in claim 1. The amplification products were detected by fluorescence quantitative PCR, and the resistance of rice to root-knot nematodes of the Poaceae family was analyzed by the detection results. When the 2902A test result is blue, it is determined that the rice does not contain the root-knot nematode resistance site qMg11-1; When the 2902A test result is red, it is determined that the rice contains the root-knot nematode resistance site qMg11-1; When the 2902A test result is green, the material to be tested contains the heterozygous root-knot nematode resistance site qMg11-1 of the grass family; The rice variety is either Rice 685 or Minghui 63.

5. The method for detecting resistance to root-knot nematodes in rice according to claim 4, characterized in that, The PCR amplification reaction system was 10 μL, as detailed below: 5 μL of FLU-ARMS 2× PCR Mix, 2 μL of genomic DNA, 0.75 μL of Primer mix, and 2.25 μL of ddH2O; the concentration ratio of primers 2902A-F1, 2902A-F2, and 2902A-R in the Primer mix was 1:1:

3.

6. The method for detecting resistance to root-knot nematodes in rice according to claim 4, characterized in that, The PCR amplification reaction procedure is as follows: Pre-denaturation at 95 ℃ for 5 min; amplification at 95 ℃ for 5 s, 58 ℃ for 20 s for 40 cycles; plate reading at 30 ℃ for 30 s for 1 cycle.

7. The application of the kit according to claim 3 or the method according to any one of claims 4-6 in the detection of resistance to root-knot nematodes in rice, wherein the KASP molecular marker primer set of the tightly linked molecular marker 2902A according to claim 1 is used to amplify DNA by PCR, and the amplification product is detected by fluorescence quantitative PCR. The judgment criteria are as follows: When the 2902A test result is blue, it is determined that the rice does not contain the root-knot nematode resistance site qMg11-1; When the 2902A test result is red, it is determined that the rice contains the root-knot nematode resistance site qMg11-1; When the 2902A test result is green, the material to be tested contains the heterozygous root-knot nematode resistance site qMg11-1 of the grass family; The rice variety is either Rice 685 or Minghui 63.

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