Early-stage efficient identification method for clubroot resistance of rape and application of early-stage efficient identification method

Abstract

The invention belongs to the technical field of plant disease-resistant molecule identification, and particularly discloses an early identification method of rape clubroot resistance. The method comprises the following steps: planting aseptic seedlings of to-be-detected rape, known clubroot-resistant rape and clubroot-susceptible rape, inoculating plasmodiophora brassicae to soil at the root of the rape in the seedling stage, extracting DNA of root tissues of the to-be-detected rape and performing qPCR detection on specific genes of the plasmodiophora brassicae on the 5th day after inoculating the plasmodiophora brassicae, taking the known clubroot-resistant rape as a control group, and performing quantitative PCR detection on the specific genes of the plasmodiophora brassicae. Calculating the relative content of the gene, and identifying the anti-susceptibility of the rape to be detected to clubroot according to the relative content of the gene. The plasmodiophora brassicae DNA is taken as a detection target for the first time, pathogens can be found earlier through DNA level detection compared with RNA level detection, DNA serving as the detection target is more reliable than RNA and has high stability, and therefore the requirement for early-stage accurate identification is met.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance molecular identification, and particularly relates to an early and efficient identification method for rapeseed root knot fungus disease resistance and an application thereof. Background Art

[0002] Clubroot is a disease caused by a protist, Plasmodiophora brassicae Woron, which is a specialized living parasite and causes great harm to cruciferous plants. The roots of plants infected with clubroot will swell abnormally, affecting the absorption of nutrients and water by the root tissue. Severely infected plants may show symptoms of water shortage and even cause the plant to die. Field test results show that clubroot can cause a 30% reduction in rapeseed yield, and in severe cases, yield losses can be as high as 80% to 90%. Over the years, this soil-borne disease has led to a decline in the yield and quality of cruciferous crops worldwide (Dixon G R. The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. J Plant Growth Regul., 2009, 28: 194-202.). Under appropriate conditions, the dormant spores of clubroot can survive in the soil for up to 17 years (HASAN J. Clubroot in Brassica: recent advances in genomics, breeding, and disease management [J]. Genome, 2021, 64 (8): 735-760.). During this period, contaminated soil will no longer be suitable for growing cruciferous crops. my country is the world's largest producer of Brassica crops (Qiu Cuijin. Analysis of World Brassica Crop Production and Trade [J]. World Agriculture, 2014 (5): 111-118, 127.). Since 1936, clubroot was first reported in Taiwan Province, my country, and then spread throughout my country. At present, clubroot has been found in at least 20 provinces in China, seriously affecting the yield and quality of cruciferous crops (Peng Lisha. Research progress on biocontrol bacteria for clubroot in cruciferous crops [J]. Plant Protection, 2015, 41 (4): 16-22.). This disease has seriously threatened the production of rapeseed and other cruciferous crops in major producing areas of my country, causing huge losses to agricultural economic development and farmers' income.

[0003] Many scholars at home and abroad have conducted a lot of research on the detection of root-knot fungi, especially in the resistance mechanism of plants to root-knot fungi and its breeding and utilization. These studies are inseparable from the screening and identification of disease-resistant germplasm resources. The early identification of disease resistance is of great significance for the comprehensive and in-depth research on root-knot fungi and the acceleration of plant breeding for resistance to root-knot fungi because it can effectively reduce labor and space costs and realize the advantages of high-throughput and large-scale screening of germplasm resources resistant to root-knot fungi under precisely controllable environmental conditions. In the study of rapeseed resistance to root-knot fungi, traditional methods mainly rely on root phenotypic analysis. Fedptova (1933) first observed the dormant spores of root-knot fungi through a microscope. This technology was subsequently further improved by Melbile (1952) and Hawken (1967) and developed into a plant induction method. This method involves planting the host plant in the soil to be tested and observing the symptoms of the host after 30 to 40 days. However, this method is time-consuming and requires a lot of manpower and culture space, which is not conducive to the high-throughput screening of germplasm resources resistant to root-knot fungi. In order to shorten the observation time and improve the identification efficiency, the researchers introduced fluorescent staining technology. By using the lipophilic dye Nile Red to stain the root knot fungus, its lipid droplet structure can be clearly observed under a fluorescence microscope. In addition, DAPI is a fluorescent dye that can strongly bind to DNA. The researchers used Nile Red in combination with DAPI to stain the cortical cells of resistant and susceptible materials. Under a fluorescence microscope, this combined staining can help distinguish materials infected by root knot fungi from uninfected materials, thereby judging the resistance and susceptibility of host plants earlier. This staining technology can shorten the time for determining resistant and susceptible materials to within 16 days after inoculation, but this method still requires high labor and space costs, and microscopic observation requires high experience of technicians, and the sample processing and observation process is also very time-consuming. With the development of molecular biology technology, the researchers screened out genes that were significantly upregulated in susceptible materials through transcriptome analysis 20 days after inoculation, and further verified them through qRT-PCR technology, and finally identified 4 marker genes. The expression levels of these marker genes in susceptible materials are significantly higher than those in resistant materials, and they can be used as molecular markers to determine the resistance and susceptibility of materials (Liu Yan, Establishment of an Early Detection System for Rapeseed Clubroot and Preliminary Identification of Clubroot Effector Factors [D], Huazhong Agricultural University, 2017). However, this technology is easily affected by environmental factors, resulting in fluctuations in test results. In addition, this technology still has problems such as long time and high space cost, which makes this method have certain limitations in practical applications.

[0004] In view of the above-mentioned defects in the early identification of plant resistance to root knot fungi, it is necessary to construct an efficient and stable early identification method for root knot fungi resistance. This is of great significance for deepening the basic research on root knot fungi, accelerating the breeding process of rapeseed resistance to root knot disease, and ensuring the safety and stability of my country's agricultural production. Summary of the invention

[0005] The present invention aims to overcome the shortcomings of the prior art by extracting DNA from rapeseed roots and using fluorescent quantitative PCR technology to detect the colonization ability of root knot fungi in rapeseed roots, and strive to establish an accurate method for identifying early infection of root knot fungi. This method will provide a convenient and efficient detection method for the in-depth development of basic research on root knot fungi and the cultivation of disease-resistant varieties.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The early identification method of rapeseed clubroot resistance comprises the following steps:

[0008] (1) After planting sterile seedlings of the rapeseed to be tested and the rapeseed known to be resistant to clubroot and susceptible to clubroot, the soil around the roots of the rapeseed was inoculated with clubroot bacteria at the seedling stage;

[0009] (2) On the 5th day after inoculation with root knot fungi, DNA was extracted from the root tissue of the rapeseed to be tested and the root knot fungi-specific gene was detected by qPCR, with rapeseed known to be resistant to root knot fungi as the control group and the internal reference gene derived from rapeseed, and the relative content of the gene was calculated;

[0010] (3) identifying the resistance of the tested rapeseed to clubroot disease based on the relative content of the gene;

[0011] Specifically, if the relative content of the root-knot fungus gene of the rapeseed to be tested is less than 1.5 times that of the known rapeseed resistant to root-knot, the rapeseed to be tested is identified as a variety resistant to root-knot; if the relative content of the root-knot fungus gene of the rapeseed to be tested is greater than 2 / 3 of that of the known rapeseed susceptible to root-knot, the rapeseed to be tested is identified as a variety susceptible to root-knot.

[0012] Preferably, the root knotweed bacteria-specific gene is PbActin, and the internal reference gene is BnActin.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] (1) For the first time, root knot fungus DNA was used as the detection target. Compared with RNA level detection, DNA level detection can detect pathogens earlier, thus meeting the needs of early identification.

[0015] (2) Using root knot fungus DNA as a detection target is more reliable than RNA, because RNA is easily affected by the external environment and has poor stability, while DNA has high stability and can meet the requirements of accurate identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The technical roadmap of the present invention.

[0017] Figure 2 :Solubility curves of primer amplification products of PbActin gene from Rhizoctonia solani.

[0018] Figure 3 : Solubility curve of the products amplified by primers of rapeseed BnActin gene.

[0019] Figure 4 : DNA qPCR detection results of PbActin gene at different times after root inoculation with root-knot fungus in NZ66 and Huashuang 5R.

[0020] Figure 5 : DNA qPCR detection results of PbActin gene in roots of NZ66, Huashuang 5R, varieties A and B at different times after inoculation of root knotweed fungi.

[0021] Figure 6 : Microscopic observation results of 1 cm area above and below the hypocotyls of NZ66, Huashuang 5R, varieties A and B after inoculation with root-knot fungi.

[0022] Figure 7 :Measurement results of the stem segment diameter at the root base of NZ66, Huashuang 5R, varieties A and B after inoculation with root knot fungi (unit: mm)

[0023] Figure 8 : Root phenotypes of NZ66, Huashuang 5R, varieties A and B 32 days after inoculation with root-knot fungi. DETAILED DESCRIPTION

[0024] Example 1: An early and efficient identification method for rapeseed clubroot resistance.

[0025] 1. Seed disinfection

[0026] First, select the seeds of the full clubroot-resistant rapeseed Huashuang 5R (conventional variety, but the implementation of the present invention is not limited to this variety) and the susceptible rapeseed variety NZ66 (laboratory self-bred variety, but the implementation of the present invention is not limited to this variety), rinse with sterile water 3-5 times, place in 75% alcohol for immersion and disinfection for 30s, and then rinse with sterile water 3-5 times. Then soak in 25% 84 disinfectant (effective chlorine content is 5%-6.5%) for about 8 minutes, and rinse with sterile water 3-5 times. Use sterile filter paper to absorb the moisture on the surface of the seeds, and then inoculate the rapeseed seeds on the solid culture medium of MS+3% sucrose (pH is 5.8), light 1200lx, and the seedlings are cultured in a growth box under the conditions of 25°C, 16 hours of light and 20°C, 8 hours of darkness.

[0027] 2. Rapeseed Seedling Cultivation

[0028] When the rapeseed sterile seedlings grew two cotyledons, they were transplanted into mixed soil (nutrient soil: vermiculite = 2:1), irrigated twice a week with 1% Huaduoduo solution, and then continued with conventional cultivation and management.

[0029] 3. Preparation of dormant spore suspension of root knotweed

[0030] The preparation method of the root-knot fungus spore solution is referenced in the literature (Schwelm A, et al. The Plasmodiophorabrassicae genome reveals insights in its life cycle and ancestry of chitinsynthases. Sci Rep, 2015, 5: 11153). First, the diseased roots of rapeseed infected with root-knot fungus were cleaned, the surface rotten tissue was removed, treated with 70% ethanol for 5 minutes, and then soaked in 10% hydrogen peroxide solution for 2 hours, and then rinsed with sterile water 5 times. Then the swollen roots were cut into small pieces, placed in sterile water and homogenized in a homogenizer for 4 minutes. After homogenization, the plant debris was filtered out through 8 layers of gauze. The filtrate was centrifuged at 2500×g for 10 minutes, the supernatant was discarded, the precipitate was suspended with sterile water, and the washing was repeated 5 times. Then suspend the precipitate in 10 ml of 50% (w / v) sucrose solution, centrifuge at 2500×g for 15 minutes, carefully transfer the supernatant containing dormant spores to another sterile centrifuge tube, add an equal amount of sterile water and mix thoroughly, centrifuge at 2500×g for 15 minutes, discard the supernatant, suspend the dormant spore precipitate of root knot fungus with sterile water, repeat this step 5 times to completely remove the sucrose residue. Finally, count the concentration of dormant spores of root knot fungus by hemocytometer.

[0031] 4. Inoculation of Brassica rapa

[0032] A small hole was dug in the soil near the roots of rapeseed to expose the root tissue, and then a suspension of 3×108 spores / L of root knotweed fungi was injected into the soil near the roots of rapeseed using a pipette, with 2 mL inoculated per rapeseed plant. After inoculation, the roots were covered with soil and no watering was performed within two days after inoculation.

[0033] 5. DNA Extraction from Rapeseed Root Tissue

[0034] Four days after inoculation, three rapeseed plants with the same growth were selected from Huashuang 5R and NZ66 for sampling. After the rapeseed roots were cleaned, the root tissue was placed in a mortar and liquid nitrogen was added to fully grind into fine powder, then the fine powder was placed in a 2ml centrifuge tube, and an appropriate amount of lysis solution was added for treatment. After a series of centrifugation, washing and DNA precipitation steps, a purified DNA sample was finally obtained. DNA extraction used the DNA extraction kit provided by Beijing Qingke Biotechnology Co., Ltd., and the specific extraction steps refer to the kit instructions.

[0035] 6.qPCR detection

[0036] qPCR reaction kit was used to analyze the PbActin gene of root knot fungus in rapeseed roots (the reaction kit was purchased from Applied Biological Materials Inc, Blastaq TM 2×qPCR MasterMix Kit). Primer synthesis was completed by Shanghai Shenggong Biotechnology Service Co., Ltd. The target gene primers PbActinF: 5'-GGGACATCACCGACTACCTG-3', PbActinR: 5'-ACTGCTCCGAGTTGGACATC-3'; the reference gene primers BnActin-F: 5'-TCTTCCTCACGCTATCCTCCG-3', BnActin-R: 5'-AGCCGTCTCCAGCTCTTGC-3'. The reaction system was: DNA (about 50ng) 0.4μL, 2×SYBR buffer 5μL, left and right primers (10μM) 0.25μL each, ddH2O 4.3μL, and the total reaction volume was 10μL. The reaction program was 95℃ pre-denaturation for 3min, and entered 40 cycles, each cycle including 95℃ denaturation for 15sec and 58℃ annealing for 1min. After the reaction, the specificity of amplification was evaluated by melting curve. The melting curve results showed that the amplified product was a single peak ( Figure 2 and Figure 3 ), indicating that the primers have good amplification specificity. -△△Ct The relative content of PbActin gene in the root tissue of rapeseed at different times (5th to 13th day) after inoculation with root knot fungi was calculated by this method, and the disease-resistant rapeseed variety (NZ66) on the fourth day was used as the control group. Figure 4 As shown in the figure, on the fifth day of inoculation, the relative content of PbActin gene between clubroot-resistant varieties and susceptible varieties was significantly different. This indicates that the fifth day is the earliest time point when clubroot-resistant materials can be accurately distinguished from susceptible materials.

[0037] Example 2: Application of an early and efficient identification method for rapeseed clubroot resistance

[0038] To further verify the reliability of the early identification method for rapeseed clubroot resistance, the inventors selected Huashuang 5R, NZ66, and two rapeseed varieties A and B with unknown resistance and susceptibility. The rapeseed culture and clubroot inoculation conditions were the same as in the previous experiment. By testing these four varieties, the applicability of this method in unknown resistance and susceptibility materials was evaluated, thereby determining its effectiveness in actual research.

[0039] 1. Sampling of rapeseed

[0040] On the fourth day after the inoculation of root-knot fungi, three plants with similar growth conditions were selected from the four rape varieties for sampling every day to ensure the uniformity and comparability of the data. First, the diameter of the stem segment at the base of the rape root was measured and recorded, because the reproduction of root-knot fungi would cause the stem segment at the base of the root to thicken, and then the average value of each variety was calculated. The measurement results are shown in Figure 7 The results showed that the stem diameter of rapeseed varieties A and NZ66 increased significantly faster than that of varieties B and Huashuang 5R. Subsequently, 1 cm long root tissue was cut from 1 cm above and below the hypocotyl of rapeseed and temporarily stored in 2 mL of FAA fixative for subsequent microscopic observation. The remaining root tissue was used for DNA extraction.

[0041] 2. Microscope Observation

[0042] The root samples were placed in a 0.1% toluidine blue solution (purchased from solarbio) for 15 seconds and then placed on a glass slide. 20 μL of sterile double distilled water was pipetted and dropped onto the sample surface, followed by a coverslip and observation under a microscope (Leica DM4B) at 400 times magnification. Figure 6 As shown in the figure, it can be clearly seen that under microscope observation, the four rape varieties showed obvious differences in disease resistance on the 14th day after inoculation. Varieties A and NZ66 had significantly more root knot protoplasm masses in their roots, while varieties B and Huashuang 5R had fewer root knot protoplasm masses in their roots. Therefore, based on the results of root microscopic morphology observation, rape varieties A and NZ66 were identified as susceptible materials to clubroot, while rape varieties B and Huashuang 5R were identified as resistant materials to clubroot.

[0043] 3. DNA Extraction and qPCR

[0044] The steps of DNA extraction and qPCR detection of root tissues of rapeseed varieties A and B were consistent with the previous experiment, using the same extraction method and detection process. The specific operations included: extracting DNA from root tissues and detecting the relative content of PbActin gene of root knot fungus by qPCR. In this experiment, Huashuang 5R was used as the control group to calculate the relative content of PbActin gene of varieties A and B. The results are shown in Figure 5As described above, the relative content of PbActin gene in rapeseed variety A was significantly higher than that in variety B from the 5th day after inoculation. According to the molecular identification results, A was judged to be a rapeseed variety susceptible to clubroot disease, and B was a rapeseed variety resistant to clubroot disease.

[0045] 4. Root phenotype observation

[0046] On the 35th day after inoculation with clubroot, the roots of rape varieties A and B were photographed to observe their swelling, with an emphasis on recording the size and distribution of the tumors, in order to assess the impact of clubroot on the two varieties and the difference in resistance and susceptibility. Figure 8 As shown in the figure, multiple nodules appeared on the roots of variety A, and the root tissue was swollen and deformed, with tumors widely distributed; while variety B had no obvious nodules and normal root structure. This intuitively shows that variety A is susceptible to clubroot, while variety B is resistant to clubroot.

[0047] In summary, through experimental calculation and result verification, the 5th day can be used as the earliest time point to distinguish between clubroot-resistant materials and susceptible materials. As time goes by, the gap in the root-root fungus-specific DNA qPCR detection results between resistant and susceptible materials gradually widens, further verifying the efficiency and accuracy of the method of the present invention in identifying clubroot resistance.

[0048] Compared with traditional methods such as microscopic observation, measurement of stem segment diameter at the root base, and phenotypic observation 30 days after inoculation, this method shows significant advantages in the early detection and quantitative analysis of clubroot disease resistance. Through efficient early identification methods, it is possible to capture differences in clubroot disease resistance more quickly and sensitively, providing an accurate early screening approach for disease-resistant rapeseed breeding and significantly shortening the identification cycle. In addition, this method effectively reduces labor and space costs, and achieves high-throughput, large-scale screening of clubroot disease-resistant germplasm resources under precisely controllable environmental conditions, providing strong support for the screening of disease-resistant resources and the acceleration of the breeding process.

Claims

1. A method for early identification of rapeseed clubroot resistance, characterized in that: The following steps are involved: (1) After planting sterile seedlings of the rapeseed to be tested and the rapeseed known to be resistant to clubroot and susceptible to clubroot, the soil around the roots of the rapeseed was inoculated with clubroot bacteria at the seedling stage; (2) On the 5th day after inoculation with root knot fungi, DNA was extracted from the root tissue of the rapeseed to be tested and the root knot fungi-specific gene was detected by qPCR. Rapeseed with known resistance to root knot fungi was used as a control group. The internal reference gene was derived from rapeseed, and the relative content of the gene was calculated; (3) Identifying the resistance of the tested rapeseed to clubroot disease based on the relative content of the gene.

2. The method according to claim 1, characterized in that If the relative content of the root-knot fungus gene of the rapeseed to be tested is less than 1.5 times that of the known rapeseed resistant to root-knot, the rapeseed to be tested is identified as a club-knot resistant variety; if the relative content of the root-knot fungus gene of the rapeseed to be tested is greater than 2 / 3 of that of the known rapeseed susceptible to root-knot, the rapeseed to be tested is identified as a club-knot susceptible variety.

3. The method according to claim 1, characterized in that The root knot fungus specific gene is PbActin, and the internal reference gene is BnActin.

4. The method according to claim 3, characterized in that The primers of PbActin are 5'-GGGACATCACCGACTACCTG-3', 5'-ACTGCTCCGAGTTGGACATC-3'.

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