Haplotype marker tightly linked to the major QTL for resistance to rice black-streaked dwarf virus on chromosome 6

Abstract

The application provides a haplotype marker of a main-effect QTL site of rice black-streaked dwarf disease resistance, wherein the bases at the sites 914797, 965164 and 1149209 of rice chromosome 6 are A, G and T respectively. A related main-effect QTL site of rice black-streaked dwarf disease resistance is provided, which is located on the chromosome 6 of rice. Related applications are also provided. The haplotype marker of the main-effect QTL site of rice black-streaked dwarf disease resistance can detect, predict and effectively select the resistance of rice black-streaked dwarf disease, and can be used for molecular marker assisted breeding of rice with high resistance to black-streaked dwarf disease, accelerating the process of rice disease resistance breeding. The design is ingenious, not affected by the environment, and suitable for large-scale popularization and application.

Description

Technical fields:

[0001] This invention relates to a haplotype marker Hap1 on rice chromosome 6 that is closely linked to a major QTL for resistance to rice black-streaked dwarf disease. It can be applied to marker-assisted breeding and belongs to the fields of rice disease resistance breeding and molecular biology. Background technology:

[0002] Rice black-streaked dwarf virus (RBSDV) causes a severe viral disease that impairs rice plant development, reduces seed setting rate, and causes significant yield losses. This disease caused widespread outbreaks in North and East China during the 1960s and late 1990s. RBSDV can also infect other crops such as corn, wheat, and barley, making it one of the most destructive crop viruses globally. To date, there are no effective chemical agents to control this viral disease. Utilizing the inherent resistance of rice varieties is the most economical and effective method for controlling viral diseases.

[0003] Rice resistance to RBSDV is a complex trait controlled by multiple genes. Current research on the genetics of rice black-streaked dwarf virus resistance only provides preliminary mapping results. More than 30 QTLs are associated with RBSDV resistance, distributed across all chromosomes except chromosome 12, indicating that differentiated RBSDV resistance mechanisms may exist among rice resources from different sources, and that the genetic mechanisms are quite complex. A thorough understanding of its molecular genetic mechanisms is fundamental to efficient and accurate molecular breeding. However, there are currently no reports of cloning and breeding applications of RBSDV resistance genes, which seriously hinders the progress of rice resistance breeding against RBSDV.

[0004] Field identification of rice black-streaked dwarf virus (RBSDV) in severely affected areas is convenient and quick, but it is easily affected by factors such as environmental conditions, annual and regional inconsistencies in the occurrence (quantity and virus carrying rate) of planthoppers, leading to significant differences in resistance identification results among different studies. Furthermore, since planthoppers also transmit rice stripe virus (RSV), they can interfere with field identification results. Artificial inoculation identification, conducted under controlled conditions, is a resistance identification method that can effectively overcome the problems that are difficult to avoid in field identification when evaluating the resistance level of rice varieties to RBSDV. However, because RBSDV is not transmitted through planthopper eggs, this method is relatively time-consuming and labor-intensive. Therefore, resistance identification has become a significant factor restricting the breeding of rice RBSDV resistance.

[0005] Molecular marker-assisted breeding technology can effectively solve the above problems. By using rice varieties with different resistance from different countries and regions, haplotype markers closely linked to the major resistance QTLs on chromosome 6 of rice can be obtained through association analysis. These markers can then be used to screen rice germplasm resources at the seedling stage, effectively selecting resistant germplasm and eliminating susceptible germplasm, thus greatly improving breeding efficiency while saving costs. Summary of the Invention:

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a haplotype marker that can effectively screen for genes that resist rice black-streaked dwarf disease and a method for using the marker.

[0007] This invention utilizes 509 rice germplasm resources from different countries and regions with varying resistance to rice black-streaked dwarf disease. Through association analysis between SNP microarray data containing a 700Kb locus and disease resistance identification results, the major QTL for rice black-streaked dwarf disease resistance on chromosome 6 was obtained. By comparing the sequence information of different rice varieties at this locus, a haplotype marker Hap1 closely linked to rice black-streaked dwarf disease resistance was developed, which can be applied to assisted selection breeding of rice varieties resistant to rice black-streaked dwarf disease.

[0008] The haplotype marker Hap1 on chromosome 6, which is closely linked to the major QTL for resistance to rice black-streaked dwarf disease, provided in this invention, was obtained through the following method:

[0009] 1) Collected 509 rice germplasm resources from different countries and regions with different resistance to black-streaked dwarf disease, and identified the resistance of each rice germplasm to black-streaked dwarf disease by combining natural disease occurrence and artificial inoculation.

[0010] 2) DNA was extracted from 509 rice germplasm resources using the CTAB method;

[0011] 3) The QTL for resistance to black-streaked dwarf disease was located through genome-wide association analysis, and the major QTL was located in a 400 kb interval of 0.8-1.2 Mb on chromosome 6;

[0012] 4) Resequencing analysis was used to identify sequence differences in the 400kb region of 222 rice germplasm resources. The correlation between all SNP site combinations in this region and disease resistance was analyzed, and the haplotype marker Hap1, which is closely related to disease resistance, was identified.

[0013] Applications of haplotype markers on chromosome 6 that are closely associated with major QTLs of resistance to rice black-streaked dwarf virus include: predicting the resistance of rice black-streaked dwarf virus by detecting the haplotype marker Hap1 on chromosome 6 of rice varieties.

[0014] This invention overcomes the shortcomings of conventional breeding methods, such as poor stability and repeatability in the identification of rice black-streaked dwarf disease resistance, as well as the time and effort required. Its results can simplify the selection method and improve breeding efficiency, thereby accelerating the breeding process of disease-resistant varieties. Attached image description:

[0015] Figure 1 The results of association analysis using SNP chip data combined with natural disease and artificial inoculation identification of 509 rice germplasm resources.

[0016] Figure 2 Haplotype markers within the 400kb interval of the major QTL for resistance to black-streaked dwarf disease on chromosome 6.

[0017] Figure 3 The correlation between the haplotype marker Hap1 and the disease index of rice black-streaked dwarf disease in 222 rice germplasm resources. Detailed implementation method:

[0018] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0019] Example 1: Obtaining haplotype markers closely linked to major QTLs of resistance to rice black-streaked dwarf disease 1. Plant materials

[0020] 509 rice materials, including indica rice, japonica rice, Aus rice, and aromatic rice, were selected from rice populations in 72 countries or regions for the identification of resistance to rice black-streaked dwarf disease.

[0021] 2. Source of the virus and inoculation

[0022] (1) Rice black-streaked dwarf virus was collected from Kaifeng, Henan Province, Jianhu, Jiangsu Province, Nanjing, Jiangsu Province, and other places. Suspected diseased plants of rice black-streaked dwarf virus with 4-5 leaf age were collected and detected by RT-PCR using RBSDV-specific primers. RBSDV-positive plants were artificially inoculated for identification. After confirmation by testing, they were transplanted to the experimental field of our laboratory to preserve the virus source for feeding experiments.

[0023] (2) Adult planthoppers were obtained from laboratory-preserved populations and reared on RBSDV-positive rice plants for 7 days. The planthoppers were then transferred to healthy Wuyujing No. 3 rice seedlings for 8 days to allow RBSDV to circulate within the planthoppers. The virus-carrying rate of the planthopper population was determined by dot-ELISA.

[0024] 3. Evaluation of resistance to rice black-streaked dwarf disease

[0025] In 2015 and 2016, rice black-streaked dwarf virus (RSV) was prevalent in Kaifeng City, Henan Province. 509 rice varieties were planted in the area to allow natural infection. To ensure sufficient insects carrying the virus, fields in the surrounding wheat fields that had previously experienced wheat green dwarf virus (also caused by RBSDV) were selected as rice seedbeds. Rice varieties were sown two weeks before wheat harvest. One month later, the seedlings were transplanted to rice paddies in Jurong, Jiangsu Province. No insecticides or antiviral drugs were applied before the onset of symptoms, and cultivation and management were the same as normal field practices. One month after transplanting, the incidence of RSV was recorded. Compared with healthy plants, diseased plants exhibited significantly more severe stunting, stiff, dark green leaves, and waxy white raised areas on the undersides of leaves, leaf sheaths, and stems. Each variety or line was replicated three times. The average of the replicates was used as the phenotypic value. The resistance of plants to rice black-streaked dwarf virus was evaluated using the incidence rate (number of infected plants / total number of plants × 100%) as an indicator.

[0026] Artificial inoculation experiments were conducted at the Jiangsu Academy of Agricultural Sciences. Rice seeds were sown in 500ml beakers. At the 1.5-leaf stage, based on the virus-carrying rate of planthoppers, two infected planthoppers were inoculated per rice seedling for 48 hours. After removing the planthoppers, the seedlings were transplanted to the cement pond experimental area of ​​the Jiangsu Academy of Agricultural Sciences. No pesticides or antiviral drugs were sprayed during the rice's growth period, and other cultivation and management were carried out according to standard field management practices. The investigation and data processing methods were the same as those used in the field natural disease identification experiment.

[0027] 4. DNA extraction from rice plants (CTAB method)

[0028] 1) Weigh 500mg of rice leaves into a 2.0mL Eppendorf tube, cool the centrifuge tube in liquid nitrogen, fill it with liquid nitrogen and then quickly remove it and grind it into powder with a grinding rod.

[0029] 2) Incubate in a water bath at 65℃ for 0.5 hours, shaking once every 10 minutes;

[0030] 3) Centrifuge at 12,000 rpm for 10 min at 4℃ and collect the upper aqueous phase;

[0031] 4) Add 900 μL of chloroform:isoamyl alcohol solution (24:1), mix thoroughly and shake until the solution color changes from green to white;

[0032] 5) Centrifuge at 12,000 rpm for 10 min at 4℃ and collect the upper aqueous phase;

[0033] 6) Add an equal volume of isopropanol (or 2 volumes of anhydrous ethanol), let stand at -20℃ for 20-30 minutes to precipitate flocculent DNA;

[0034] 7) Centrifuge at 12,000 rpm for 10 min at 4℃, discard the supernatant, add 1 mL of 70% ethanol to wash, centrifuge at 12,000 rpm for 5 min at 4℃, discard the supernatant and blot dry with filter paper, and place on a clean bench to air dry.

[0035] 8) Dissolve the DNA in 30 μL of deionized water and store at 4°C for later use.

[0036] The OD value and concentration were measured using the Eppendorf BioPhotometer Plus nucleic acid protein analyzer, and the DNA of each sample was diluted to 20 ng / μl for later use.

[0037] 5. Genome-wide association analysis

[0038] Genotyping of rice plants was analyzed using a 700kb microarray based on the Illumina platform. GWAS analysis was performed using GAPIT V2 based on criteria of missing data less than 15% and minor allele frequency (MAF) > 0.05. A mixed linear model with a kinship matrix was used, and PCA was performed in GAPIT. Manhattan plots were generated using the CMR software package. The rice genome sequence version MSU V7.0 was used as the analytical reference.

[0039] Using 700kb SNP data, GWAS was employed to mark and map phenotypic data from natural and artificial inoculation in the field (P < 10). -4 A major-effect QTL, qRBSDV6, was mapped on chromosome 6 of the rice variety VANDANA, within the 0.8-1.2 Mb region of chromosome 6. Figure 1 The P-value reached 3.24E-06.

[0040] 6. Haplotype analysis

[0041] Resequencing analysis of 222 rice germplasm resources in the qRBSDV6 region revealed three single nucleotide polymorphism (SNP) sites at positions 914797, 965164, and 1149209 on chromosome 6, constituting four haplotypes: Hap1, Hap2, Hap3, and Hap4. Figure 2 Hap1 is closely associated with resistance to black-streaked dwarf disease.

[0042] Example 2: Application of haplotype markers on chromosome 6 closely linked to QTLs for resistance to rice black-streaked dwarf disease in 222 rice germplasm resources.

[0043] The haplotype marker Hap1, obtained from chromosome 6 and tightly linked to the QTL for resistance to rice black-streaked dwarf virus (RSV), was used to analyze the RSV resistance of 222 rice germplasm resources. DNA was extracted from each individual plant and resequencing was performed. Bases at positions 914797, 965164, and 1149209 on chromosome 6 were analyzed. Varieties with the haplotype marker Hap1 showed significantly higher resistance than those with other haplotypes. Figure 3 ).

[0044] The above embodiments do not limit the invention in any way.

Claims

1. A method for detecting resistance to rice black-streaked dwarf virus based on haplotype markers, characterized in that, Includes the following steps: (1) The haplotype markers of the major QTL loci for resistance to rice black-streaked dwarf disease include the bases at loci 914797, 965164 and 1149209 on chromosome 6, which are A, G and T, respectively; (2) Extract genomic DNA from the rice to be tested and re-sequencing the rice genome; (3) Using the rice genome sequence version MSU V7.0 as the analysis reference, if the bases at positions 914797, 965164 and 1149209 on chromosome 6 of the rice to be tested are A, G and T respectively, then the rice to be tested is resistant to rice black-streaked dwarf disease; otherwise, the rice to be tested is not resistant to rice black-streaked dwarf disease.

2. The method for detecting resistance to rice black-streaked dwarf disease according to claim 1 is applied to the detection of the level of resistance to rice black-streaked dwarf disease.

3. The application of the method for detecting rice black-streaked dwarf disease resistance according to claim 1 in predicting the level of rice black-streaked dwarf disease resistance.

4. The application of the method for detecting rice black-streaked dwarf disease resistance according to claim 1 in selecting rice plants based on their resistance levels to rice black-streaked dwarf disease.

5. The application of the method for detecting rice black-streaked dwarf disease resistance according to claim 1 in rice black-streaked dwarf disease resistance breeding.

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