Rice whole genome molecular marker combination and application thereof

Abstract

The present application relates to the technical field of plant breeding, and particularly relates to a rice whole genome molecular marker combination and application thereof. The molecular marker combination comprises molecular markers with sequence numbers 1-329 shown in table 1 and molecular markers with sequence numbers 1-1058 shown in table 2. The present application carries out systematic screening on functional gene and non-functional gene related SNP sites of rice, and obtains a rice whole genome molecular marker detection system, which can be applied to gene typing of plants, molecular marker assisted breeding of plants, screening or identification of rice blast resistant plant plants or germplasm resources, genetic population structure analysis, kinship evaluation, dominant gene combination selection or germplasm resource diversity evaluation of plants, plant variety identification or purity detection, kinship evaluation of plants, plant variety improvement or excellent trait introduction, and preparation of molecular breeding chips of plants, and has important value in the field of plant breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a combination of whole-genome molecular markers for rice and their applications. Background Technology

[0002] Rice is one of the most important food crops, and its yield and quality are directly related to food security and sustainable agricultural development. Developing high-yielding, high-quality, disease- and pest-resistant, and stress-resistant green rice varieties has always been a core objective of rice breeding. Traditional breeding methods mainly rely on hybridization, self-pollination, backcrossing, and phenotypic selection. However, due to limitations such as significant environmental influences, difficulty in directly identifying genotypes, and long selection cycles, breeding efficiency is low, making it difficult to achieve precise improvement of target traits.

[0003] Marker-assisted selection (MAS) breeding technology provides an effective means to overcome the limitations of traditional breeding. This technology directly identifies target genotypes through molecular markers, unaffected by environmental factors and allele dominance / recessiveness, enabling precise screening of segregating populations in early generations, thus significantly improving selection efficiency and shortening the breeding cycle. Currently, MAS technology is relatively mature and has played an important role in crop disease resistance, stress resistance, quality improvement, and genetic analysis of complex traits. Through MAS technology, researchers can precisely introduce or aggregate specific target genes, such as genes for resistance to rice blast. Pi2 , Pigm or quality-related genes BADH2 , Wx This allows for the simultaneous improvement of resistance and quality traits, significantly enhancing the directionality and controllability of breeding.

[0004] However, existing rice molecular marker resources still suffer from problems such as insufficient coverage of functional sites, poor marker universality, and imperfect detection systems, making it difficult to meet the needs of multi-trait aggregation and precise genome-wide selection in commercial breeding. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a combination of whole-genome molecular markers for rice and its application.

[0006] This invention, based on functional genes in rice related to major agronomic traits such as yield, growth period, stress resistance, disease resistance, and quality, systematically screened and developed functional loci, forming a standardized whole-genome molecular marker detection system for rice. This system enables rapid identification and aggregation of dominant allelic variations in key genes, providing technical support for efficient molecular breeding, significantly improving the accuracy and efficiency of rice breeding, and possessing significant scientific value and application prospects.

[0007] In a first aspect, the present invention provides a molecular marker combination, including: molecular markers numbered 1-329 as shown in Table 1, and molecular markers numbered 1-1058 as shown in Table 2.

[0008] Tables 1 and 2 are shown below. The tables record the chromosomes on which each molecular marker is located, as well as its physical location and allelic type: Table 1 Combinations of functional gene molecular markers

[0009] Table 2 Combinations of molecular markers for non-functional genes

[0010] Secondly, this invention provides a primer pair for amplifying the aforementioned molecular marker combination. The primer pair design method of this invention can be a conventional method of this invention. Technicians can design primer pairs of different lengths (including primer pairs or KASP primer combinations) for amplifying the aforementioned molecular markers based on existing primer design rules and primer design software (such as Primer).

[0011] Thirdly, the present invention provides a probe combination for detecting the aforementioned molecular marker combination.

[0012] Fourthly, the present invention provides a kit comprising: the aforementioned molecular marker combination, or the aforementioned primer combination, or the aforementioned probe combination.

[0013] Fifthly, the present invention provides the aforementioned combination of molecular markers as targets for application in any of the following: (1) Genotyping of plants; (2) Molecular marker-assisted breeding of plants; (3) Screening or identifying plant plants or germplasm resources resistant to rice blast; (4) Analysis of plant genetic population structure, assessment of kinship, selection of dominant gene combinations or assessment of germplasm resource diversity; (5) Plant variety identification or purity testing; (6) Assessment of plant phylogenetic relationships; (7) Plant variety improvement or introduction of superior traits; (8) Prepare molecular breeding chips for plants.

[0014] In a sixth aspect, the present invention provides the use of the aforementioned primer combination, or the aforementioned probe combination, or the aforementioned kit in any of the following: (1) Genotyping of plants; (2) Molecular marker-assisted breeding of plants; (3) Screening or identifying plant plants or germplasm resources resistant to rice blast; (4) Analysis of plant genetic population structure, assessment of kinship, selection of dominant gene combinations or assessment of germplasm resource diversity; (5) Plant variety identification or purity testing; (6) Assessment of plant phylogenetic relationships; (7) Plant variety improvement or introduction of superior traits.

[0015] Furthermore, the genes include: functional genes or non-functional genes.

[0016] Furthermore, the gene is a gene related to plant yield, quality, fertility, disease and pest resistance, or stress tolerance traits; Preferably, the genes include: An-1, An-2, Gn1a, GNP1, GW6a, Gy1, HL6, HTD1, LAX1, LOX-3, NAL1, NOG1, PTB1, SGDP7, TGW6, BAT2, D2, OsTb2, TAC1, TAC3, Rc, GE, GF14g, GL3.1, GL3.2, GL3.3, GL7, GLA, GRF4, GS3, GS5, GS6, GW2, GW5, GW7, GW8, OsERF 115. OsGF14d, OsGF14h, OsLG3, OsPAO5, OsSNB, OsSPL13, qLGY, RAE2, RGA1, OsLG1, D61, GLR1, ILI3, OsbHLH174, O sPME1, OsSGR, OsTSD2, OsUGT706D1, OsOTUB1, OsSPY, sd1, SLR1, TIG1, DFOT1, OsMYB8, Ef-cd, Ehd1, Ehd4, Ghd7, G hd7.1, Ghd8, Hd1, Hd16, Hd17, Hd18, Hd3a, Hd6, Hd7, Hd9, MIR528, OsCOL4, OsHESO1, OsMADS51, OsMADS56, RCN1, R FT1, ALS, HIS1, OsACC2, SBI, OsTB1, SCM2, SCM3, Sdr4, bZIP73, COG2, COG3, COLD1, CTB2, CTB4a, HAN1, LTG1, OsbZ IP54, OsCTK1, OsGSTZ2, OsLTPL159, OsMYB2, OsSAP16, OsSEH1, qLTG3-1, qPSR10, qPSR7-2, DCA1, DRG9, DRO1, DRO T1, OsJAZ1, OsNCED2, OsPP15, qRT9, TIPS-11-9, HTH5, HTS1, OsHTAS, OsNRT2.3, qHT7, SLG1, TOGR1, TT1, TT2, TT3.1. TTL1, OsCBL10, OsTPP7, Sub1, LEA12, OsHKT1;1, Pup1, qSE3, SKC1, BOC1, BST, eIF4G, ESA1, Gm4, OsAT4c, OsGSK2, OsTBT1, OsTBT2, OsUGT706C2, Phr1, qUVR-10, Rd, psr1, qSH1, SH4, BET1, CAL1, NRAT1, OsCd1, OsHM A3, OsHMA4, OsMOT1;1, OsNRAMP5, Xa1, Xa13, Xa21, Xa23, xa25, Xa26, Xa4, xa5, Xa7, Bph14, Bph27, Bph28, B PH29, Bph3, Bph30, Bph32, Bph33, Bph6, BPH9, bsr-d1, LHCB5, OsCERK1, Pb1, Pi1-5, Pi2, Pi20, Pi35, Pi36, P i5(t), Pi56, Pi65(t), Pi9, Pia, Pib, Pi-CO39, Pid2, Pid3, Pigm, Pii, Pikh, Pit, Pita, Pizt, Ptr, Bph31, S TV11, ROD1, ALK, Badh2, qGC10, Wx, Chalk5, GFR1, WCR1, LOC_Os07g04970, OsAAP6, OsACS6, OsGluA2, pms1, S 5. One or more of the following: pms3, tms5, Rf1, Rf1b, Rf2, Rf3, Rf4, Rf6, DGS1, DPL1, DPL2, HSA1a, HSA1b, Hwi2, S28, S7, SaF, SaM, Sc, ARE1, DEP1, NGR5, NRT1.1B, OsHKT2;1, OsNLP4-OsNiR, OsNPF3.1, OsNPF6.1, OsNR2, OsTCP19, or TOND1.

[0017] Furthermore, the allelic type of the molecular marker is detected by gene sequencing, probe hybridization, or PCR amplification.

[0018] Those skilled in the art, upon learning of the contents of the functional gene molecular marker combinations in Tables 1 and 2 above, can detect their allelic types using conventional methods in the art, such as direct gene sequencing.

[0019] Furthermore, the plants include monocotyledonous plants or dicotyledonous plants; Preferably, the plant is a member of the genus *Oryza*. More preferably, the plant is rice.

[0020] The present invention has the following beneficial effects: Based on the rice genome, this invention develops a set of molecular marker combinations covering functional or non-functional genes. Detection of this molecular marker combination can rapidly detect the allelic types of functional and non-functional genes in rice germplasm materials, with stable and reliable results, simplified operation procedures, and low detection costs.

[0021] The molecular marker combination provided by this invention can achieve high-throughput detection of target genes and screening of superior combinations, effectively supporting molecular detection and aggregation of superior genes in rice breeding materials, and accelerating the breeding process of high-quality new rice varieties.

[0022] The molecular marker combination provided by this invention is compatible with the detection needs of different varieties, different ecotypes and multiple trait genes, and can be widely used in scientific breeding, commercial breeding and variety identification, providing key technical support for the precision and efficiency of rice molecular breeding. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This shows the distribution of the molecular marker combinations provided in this embodiment of the invention on rice chromosomes.

[0025] Figure 2 This is a phylogenetic tree based on molecular marker combinations provided in this embodiment of the invention; wherein, ECI: early indica rice; SCI: conventional indica rice in South China; 2LR: two-line restorer line; 3LR: three-line restorer line; 2LS: two-line male-sterile line; LCI: late indica rice; 3LM: three-line male-sterile maintainer line.

[0026] Figure 3 This is a kinship network diagram based on molecular marker combinations provided in an embodiment of the present invention.

[0027] Figure 4 The results of population genetic structure analysis provided in the embodiments of the present invention are as follows: ECI: early indica rice; SCI: conventional indica rice in South China; 2LR: two-line restorer line; 3LR: three-line restorer line; 2LS: two-line male-sterile line; LCI: late indica rice; 3LM: three-line male-sterile maintainer line.

[0028] Figure 5 The results of the genetic background analysis of the restorer line Huahui 8612 provided in this embodiment of the invention (R608) are shown. The red circles represent genes from R608.

[0029] Figure 6 This is the genetic background analysis result (Hua Zhan) of the restorer line Hua Hui 8612 provided in the embodiment of the present invention. The blue circles represent genes from Hua Zhan.

[0030] Figure 7 The results of genetic background analysis of the restorer line Huahui 8612 provided in this embodiment of the invention are shown. The red, blue and black circles represent genes, with red circles representing genes from R608 and blue circles representing genes from Huazhan.

[0031] Figure 8 The present invention provides statistical analysis results of rice germplasm resources' resistance levels to rice blast; where a is the statistical result of the number of varieties with different resistance levels; b is the statistical result of the resistance levels of different types of varieties; and c is the statistical result of the resistance levels of varieties bred in different years.

[0032] Figure 9 The following are the results of the analysis on the relationship between the number of rice blast resistance genes and resistance provided in the embodiments of the present invention: where a is the statistical result of the number of varieties carrying different numbers of disease resistance genes; b is the statistical result of the number of disease resistance genes carried by different types of varieties; c is the statistical result of the number of disease resistance genes carried by varieties bred in different years; and d is the analysis result of the relationship between the number of disease resistance genes and resistance.

[0033] Figure 10 These are the results of the effects and distribution frequencies of the resistance gene provided in the embodiments of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0036] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0037] Example 1: Development and validation of a whole-genome molecular marker system for rice This invention aims to construct a highly efficient molecular marker detection system covering the entire rice genome. It involves systematic screening and primer development of SNP sites related to functional and non-functional genes in rice, resulting in a standardized marker system that can be used for commercial breeding and scientific research. The details are as follows: 1. Materials and Methods This invention comprehensively utilizes resequencing data from rice populations both domestically and internationally. Data sources include the Rice 3K Genome Database and 1200 parental accessions sequenced independently by the Longping Academy of Sciences, totaling 5152 rice genotype data. All data underwent FastQC quality assessment, and low-quality reads and adapter sequences were removed using Trimmomatic and Cutadapt techniques to ensure the sequencing data quality met downstream analysis requirements.

[0038] After sequence processing, clean reads were aligned to the Nipponbare reference genome (IRGSP-1.0) using the BWA-MEM algorithm. SAMtools was used for sorting and indexing, and GATK HaplotypeCaller was used for genome-wide variant detection to obtain an initial set of SNP / Indel variants. Initial screening was performed based on variant quality parameters (QUAL≥30, QD≥2, FS≤60, MQ≥40), further removing sites with deletion rates >20%, minor allele frequencies (MAF) <0.05, and heterozygosity >50% to improve the stability and representativeness of subsequent marker development.

[0039] To ensure balanced genome-wide coverage, an LD pruning strategy (window 350 kb, r) was adopted. 2 <0.6) Select representative SNP loci to preserve genetic differences between populations.

[0040] 150 bp sequence fragments were extracted upstream and downstream of the target SNP site, and primers were designed in batches. The Tm value of the resulting primer sequences was controlled at 58-62℃, and specificity detection was used to ensure the absence of dimers and non-specific binding.

[0041] The total volume of the PCR reaction system is 1.6 μL, which includes 0.8 μL of DNA template (concentration 10-30 ng / μL), 0.8 μL of PCRMaster Mix, and premixed primer solution (Fam:Hex:Com = 1∶1∶2, the amount used is 1 / 36 of the reaction system).

[0042] The PCR amplification program was set as follows: pre-denaturation at 95℃ for 3 min; 10 Touchdown cycles (95℃ for 20 s, decreasing by 60 s at 65–57℃); and 28 conventional amplification cycles (95℃ for 20 s, 57℃ for 40 s). After the reaction, fluorescence signals were acquired and automatically genotyped using the LGC IntelliQube system.

[0043] 2. Results and Analysis After screening and validation using the above process, a total of 1648 sites were obtained. Further screening and validation of the marker sites resulted in 1387 sites being successfully converted into usable molecular markers (as shown in the table below; specific sites and primer lists are in Tables 1 and 2), with an overall conversion rate of 94.7%. The conversion rate for functional gene markers was 80.0%, and the conversion rate for non-functional markers was 85.6%.

[0044] Table 3. Statistical information on molecular marker classification in rice

[0045] The markers were evenly distributed across the genome, with an average density of 0.24 Mb / marker. Chromosome 1 had the highest number of markers (201, density 0.22 Mb / marker), while chromosome 12 had the lowest. Functional gene markers showed a relatively concentrated distribution on chromosomes 1, 3, and 6 (e.g., ...). Figure 1 (As shown).

[0046] The final constructed rice genome-wide molecular marker system contains 1387 high-quality markers, covering functional genes and genomic background loci. These markers are evenly distributed, highly specific, and exhibit high transformation efficiency. This system can support the precise localization of important agronomic traits in rice, the identification of varietal authenticity, and marker-assisted selection, providing efficient and reliable technical support for commercial molecular breeding of rice.

[0047] Example 2: Rice genetic population analysis based on a whole-genome molecular marker detection system This embodiment utilizes the whole-genome molecular marker combination of rice constructed in Example 1 to perform genetic population analysis on 558 representative rice breeding materials to reveal the genetic relationships and population structure among different types of materials. The genotypes of each material at the molecular markers were directly detected using gene sequencing, as detailed below: 1. Materials and Methods The materials used included 558 representative rice varieties, including early indica rice, two-line male-sterile lines, three-line maintainer lines, two-line restorer lines, three-line restorer lines, conventional indica rice in South China, and late indica rice. Genome-wide molecular markers were used for genotyping to obtain polymorphic marker data. Based on the detection results, PLINK was used to calculate genetic distances, Powermarker was used to construct phylogenetic trees and visualized using iTOL; GEMMA was used to calculate kinship coefficients and generate relationship networks in Cytoscape; ADMIXTURE was used for population structure analysis, with K values ​​set between 2 and 7, and cross-validation error was used to determine the optimal classification.

[0048] 2. Results and Analysis The results showed that the genetic structure of the 558 rice materials exhibited significant differentiation and hierarchical characteristics. Early indica rice and sterile lines formed independent branches, while maintainer lines and restorer lines showed high genetic similarity, indicating a shared genetic background during their breeding process. Kinship analysis revealed that most varieties were closely related, forming several relatively stable genetic populations, with the most significant clusters centered on varieties such as Huahui 2246, R608, and D297B. Population structure analysis further revealed that the rice materials could be divided into two main indica rice subgroups and several subgroups, corresponding to different types of breeding materials. Some varieties showed genetic mixing, reflecting gene introgression and complex genetic backgrounds among the breeding materials (e.g., ...). Figures 2-4 ).

[0049] In summary, this embodiment, through high-throughput genotyping data from the rice whole-genome molecular marker detection system and multidimensional genetic analysis methods, clarified the population structure and kinship differentiation patterns of major rice breeding types, providing a scientific basis and technical support for hybrid parent selection, genetic background analysis, and population optimization.

[0050] Example 3: Variety genetic background analysis based on rice whole-genome molecular marker detection system This embodiment uses the restorer line Huahui 8612 and its parents R608 and Huazhan as research objects. Using the rice whole-genome molecular marker detection system established in Example 1, the genomic genetic composition and functional gene inheritance were analyzed to clarify its genetic origin and the aggregation of superior traits, as detailed below: 1. Materials and Methods Genomic DNA from three samples was collected, and 236 functional gene loci in the rice whole-genome molecular marker detection system were detected using gene sequencing. The gene contribution of each parent and the genetic composition of Huahui 8612 were analyzed by allele frequency statistics and heritability ratio calculation.

[0051] 2. Results and Analysis The analysis results show (e.g.) Figures 5-7In the Hua Hui 8612 genome, R608 contributes 37.9% genetically and Hua Zhan contributes 46.7%, with its genotypic structure intermediate between the parents. Functional genotyping shows that R608 carries 86 favorable alleles and Hua Zhan carries 91, totaling 71. The superior genes unique to R608 mainly involve high-quality (… Chalk5, qGC10 ), nitrogen high efficiency ( NGR5, ARE1 ),Yield( NAL1, GL3.2, GS6 ), reproductive period ( Hd17, Ghd8 ), drought resistance ( OsJAZ1, TIPS-11-9 ) and plant type ( TIG1 Regulation; Hua Zhan's unique genes encompass regenerative capacity ( psr ), Indica and Japonica rice are compatible ( SaM Herbicide resistance () HIS1 ), resistance to rice blast ( Pib, Pi2, Pi56, Pi5(t), Pikh Insect-resistant () BPH29 ), disease resistance ( Xa4 ) and nitrogen-efficient and quality-related genes ( OsTCP19, TOND1, GS5, COG3, OsGluA2 )wait.

[0052] Ninety-one beneficial functional genes were detected in Huahui 8612, originating from the dominant alleles of R608 and Huazhan, with the R608-derived allele being a typical example. GL3.2, NAL1, OsJAZ1, NGR5, Ghd8, TIG1 and the origins of Chinese occupation psr1, SaM, BPH29, Pi2, OsTCP19, OsGluA2 Furthermore, Huahui 8612 also contains superior genes. Wx b Gene restoration Rf1b, Rf3, Rf4 Rice blast resistance gene Pita Nitrogen-efficient genes NRT1.1B, OsNLP4, OsNR2 and heat-resistant genes TT3.1 wait.

[0053] In summary, Huahui 8612 achieved effective aggregation of superior alleles from both parents through hybridization, resulting in a superior genetic background that combines traits such as stress resistance, high yield, high quality, and strong resilience. This result validates the accuracy and practicality of the whole-genome molecular marker detection system in rice variety genetic composition analysis, and can provide efficient technical support for molecular marker-assisted breeding and the improvement of superior varieties.

[0054] Example 4: Analysis of rice blast resistance characteristics based on a whole-genome molecular marker detection system This embodiment utilizes the rice whole-genome molecular marker detection system constructed in Example 1 to perform functional verification and efficacy evaluation of molecular markers related to rice blast resistance, in order to clarify the distribution characteristics of resistance genes and their correlation with phenotypic resistance, as detailed below: 1. Materials and Methods Of the 558 rice germplasm resources selected in Example 2, 441 yielded field data for rice blast resistance evaluation. Resistance identification was conducted under diseased nursery conditions and graded according to a 1-9 standard (grade 1 being highly resistant and grade 9 being highly susceptible). Genotyping of 26 rice blast resistance genes in the samples was performed using a whole-genome molecular marker detection system for rice.

[0055] 2. Results and Analysis The results showed that the distribution of resistance levels in the population was left-skewed, with a high proportion of highly resistant materials. Level 1 resistant materials accounted for 39.2% (173 accessions), and the overall resistance level gradually increased with breeding generations. Significant differences in average resistance were observed among different population types: the two-line restorer line (2LR) had an average resistance level of 2.2, making it the most resistant population overall; the average resistance level for conventional indica rice in South China (SCI) was 2.3; and the average resistance level for the three-line maintainer line (3LM) was 4.4, indicating relatively weak resistance (e.g., ...). Figure 8 ).

[0056] The number of resistance genes carried ranges from 6 to 12, with 10 being the most common. There is a significant negative correlation between the number of carried genes and the resistance level (R0). 2 =0.96), indicating that polygenic aggregation significantly enhances disease resistance. Analysis of variance results show that... Pi2 The phenotypic explanation effect was strongest (F=44.8, P<0.01), followed by Pita (F=38.4, P<0.01) and Ptr (F=26.36, P<0.01), which plays a dominant role in rice blast resistance; Pi5(t), Pi56, Pib, Pigm Isogenes also showed significant contributions (F values ​​were 11.6, 10.0, 8.5, and 8.3, respectively, P < 0.01) (e.g. Figure 9 (As shown).

[0057] Gene distribution frequency analysis showed that LHCB5, OsCERK1, Pid2, Pid3 Broad-spectrum resistance genes are widely present, with a distribution frequency exceeding 99%. Pia The gene distribution frequency reached 83.6%; while the major gene Pi2, Pita, Ptr, Pigm The distribution frequencies were 28.9%, 43.2%, 31.2%, and 8.1%, respectively. Pigm Its utilization rate is the lowest, and it has significant potential for improvement (e.g., Figure 10 (As shown).

[0058] This embodiment verifies the accuracy and applicability of the rice whole-genome molecular marker detection system in rice blast resistance gene typing, and clarifies the effects and distribution patterns of key resistance genes. This system can be widely applied to the screening of rice disease-resistant germplasm resources, resistance gene aggregation design, background reversion rate detection, and variety authenticity identification, providing efficient and accurate technical support for molecular marker-assisted breeding.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecular marker combination, characterized in that, include: Molecular markers numbered 1-329 are shown in Table 1, and molecular markers numbered 1-1058 are shown in Table 2.

2. A primer combination, characterized in that, Used to amplify the molecular marker combination of claim 1.

3. A probe assembly, characterized in that, Used for detecting the molecular marker combination of claim 1.

4. A reagent kit, characterized in that, include: The molecular marker combination of claim 1, or the primer combination of claim 2, or the probe combination of claim 3.

5. The application of the molecular marker combination of claim 1 as a target in any of the following: (1) Genotyping of plants; (2) Molecular marker-assisted breeding of plants; (3) Screening or identifying plant plants or germplasm resources resistant to rice blast; (4) Analysis of plant genetic population structure, assessment of kinship, selection of dominant gene combinations or assessment of germplasm resource diversity; (5) Plant variety identification or purity testing; (6) Assessment of plant phylogenetic relationships; (7) Plant variety improvement or introduction of superior traits; (8) Prepare molecular breeding chips for plants.

6. The use of the primer combination of claim 2, the probe combination of claim 3, or the kit of claim 4 in any of the following: (1) Genotyping of plants; (2) Molecular marker-assisted breeding of plants; (3) Screening or identifying plant plants or germplasm resources resistant to rice blast; (4) Analysis of plant genetic population structure, assessment of kinship, selection of dominant gene combinations or assessment of germplasm resource diversity; (5) Plant variety identification or purity testing; (6) Assessment of plant phylogenetic relationships; (7) Plant variety improvement or introduction of superior traits.

7. The application according to claim 5 or 6, characterized in that, The genes mentioned are those related to plant yield, quality, fertility, disease and pest resistance, or stress tolerance traits. Preferably, the genes include: An-1, An-2, Gn1a, GNP1, GW6a, Gy1, HL6, HTD1, LAX1, LOX-3, NAL1, NOG1, PTB1, SGDP7, TGW6, BAT2, D2, OsTb2, TAC1, TAC3, Rc, GE, GF14g, GL3.1, GL3.2, GL3.3, GL7, GLA, GRF4, GS3, GS5, GS6, GW2, GW5, GW7, GW8, OsERF 115. OsGF14d, OsGF14h, OsLG3, OsPAO5, OsSNB, OsSPL13, qLGY, RAE2, RGA1, OsLG1, D61, GLR1, ILI3, OsbHLH174, O sPME1, OsSGR, OsTSD2, OsUGT706D1, OsOTUB1, OsSPY, sd1, SLR1, TIG1, DFOT1, OsMYB8, Ef-cd, Ehd1, Ehd4, Ghd7, G hd7.1, Ghd8, Hd1, Hd16, Hd17, Hd18, Hd3a, Hd6, Hd7, Hd9, MIR528, OsCOL4, OsHESO1, OsMADS51, OsMADS56, RCN1, R FT1, ALS, HIS1, OsACC2, SBI, OsTB1, SCM2, SCM3, Sdr4, bZIP73, COG2, COG3, COLD1, CTB2, CTB4a, HAN1, LTG1, OsbZ IP54, OsCTK1, OsGSTZ2, OsLTPL159, OsMYB2, OsSAP16, OsSEH1, qLTG3-1, qPSR10, qPSR7-2, DCA1, DRG9, DRO1, DRO T1, OsJAZ1, OsNCED2, OsPP15, qRT9, TIPS-11-9, HTH5, HTS1, OsHTAS, OsNRT2.3, qHT7, SLG1, TOGR1, TT1, TT2, TT3.

1. TTL1, OsCBL10, OsTPP7, Sub1, LEA12, OsHKT1;1, Pup1, qSE3, SKC1, BOC1, BST, eIF4G, ESA1, Gm4, OsAT4c, OsGSK2, OsTBT1, OsTBT2, OsUGT706C2, Phr1, qUVR-10, Rd, psr1, qSH1, SH4, BET1, CAL1, NRAT1, OsCd1, OsHM A3, OsHMA4, OsMOT1;1, OsNRAMP5, Xa1, Xa13, Xa21, Xa23, xa25, Xa26, Xa4, xa5, Xa7, Bph14, Bph27, Bph28, B PH29, Bph3, Bph30, Bph32, Bph33, Bph6, BPH9, bsr-d1, LHCB5, OsCERK1, Pb1, Pi1-5, Pi2, Pi20, Pi35, Pi36, P i5(t), Pi56, Pi65(t), Pi9, Pia, Pib, Pi-CO39, Pid2, Pid3, Pigm, Pii, Pikh, Pit, Pita, Pizt, Ptr, Bph31, S TV11, ROD1, ALK, Badh2, qGC10, Wx, Chalk5, GFR1, WCR1, LOC_Os07g04970, OsAAP6, OsACS6, OsGluA2, pms1, S 5. One or more of the following: pms3, tms5, Rf1, Rf1b, Rf2, Rf3, Rf4, Rf6, DGS1, DPL1, DPL2, HSA1a, HSA1b, Hwi2, S28, S7, SaF, SaM, Sc, ARE1, DEP1, NGR5, NRT1.1B, OsHKT2;1, OsNLP4-OsNiR, OsNPF3.1, OsNPF6.1, OsNR2, OsTCP19, or TOND1.

8. The application according to any one of claims 5-7, characterized in that, The allelic type of the molecular marker was detected by gene sequencing, probe hybridization, or PCR amplification.

9. The application according to any one of claims 5-8, characterized in that, The plants include monocotyledonous plants or dicotyledonous plants; Preferably, the plant is a member of the genus *Oryza*. More preferably, the plant is rice.

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