Method for identifying verticillium wilt resistance of solanum plants

Abstract

The invention relates to the technical field of verticillium wilt resistance identification, in particular to a method for identifying verticillium wilt resistance of solanum plants. By detecting the relative expression quantity of the Ve1 gene in the solanum plant, the disease index of the solanum plant to be detected can be determined, and compared with a traditional artificial inoculation identification method, the method has the advantages of rapidness, high efficiency, time reduction and economic cost reduction. Furthermore, a stem grafting inoculation method is applied to perform verticillium wilt artificial inoculation on the to-be-detected solanum plants, and compared with a root soaking method and a root damaging method of traditional artificial inoculation, the method has the advantages that the operation steps are simplified, the illness state observation period is shortened, and the method is rapid, efficient and high in repeatability; compared with a traditional leaf inoculation method (inoculation is performed on a single leaf), the stem grafting inoculation method has the advantages that illness state grading can be performed by observing the overall leaf and stem conditions, experimental results are more scientific and reliable, and operability and repeatability are higher.

Description

Technical Field

[0001] The invention relates to the technical field of identification of Verticillium wilt resistance, and in particular to a method for identifying Verticillium wilt resistance of Solanum plants. Background Art

[0002] Eggplant Verticillium wilt is a soil-borne plant vascular disease caused by Verticillium spp. It is one of the main diseases threatening eggplant production. It is difficult to prevent and control due to the lack of effective chemical agents. It is currently one of the important diseases in agricultural production.

[0003] Due to the lack of disease-resistant resources and insufficient utilization, the breeding of eggplant varieties resistant to Verticillium wilt has progressed slowly. Studies have shown that wild eggplant resources have been formed under harsh natural environmental conditions through long-term selection, and have strong adaptability and resistance to stress, disease, and insects, and have great potential for utilization. These wild resources provide valuable germplasm materials for the origin, evolution, classification research, and disease-resistant breeding of eggplant, greatly enriching the eggplant disease-resistant gene pool, and are also excellent rootstocks used in grafted cultivation. my country has abundant wild eggplant resources, and their phenotypic traits and disease and insect resistance are quite different from those of cultivated varieties. Especially in the humid and hot areas of southern Yunnan and tropical rainforests, there are abundant wild species and wild related species. It is urgent to explore Solanum L. varieties with good resistance to Verticillium wilt.

[0004] Traditionally, eggplant resistance to Verticillium wilt is identified mainly through the root injury method or the root immersion method, which involves artificial inoculation of eggplant materials with Verticillium wilt at the seedling stage. This method is time-consuming and labor-intensive, and the results vary greatly due to different manual operations or different growth environments. The identification results have poor repeatability and low accuracy. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for identifying the resistance of Solanum to Verticillium wilt. The method provided by the present invention can quickly and efficiently classify the resistance of Solanum to Verticillium wilt, and the method is scientific and reasonable, highly repeatable and accurate.

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

[0007] The present invention provides a method for identifying resistance to Verticillium wilt in Solanum plants, comprising the following steps:

[0008] Four true leaf stage leaves of the Solanum plant to be tested are used as the test material, and the relative expression amount of the Ve1 gene in the test material is detected using a first primer pair, wherein the first primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0009] Determining the disease index of the Solanum plant to be tested according to the relative expression amount of the Ve1 gene in the tested material; when the relative expression amount of the Ve1 gene is greater than 2.0, the disease index of the Solanum plant to be tested is greater than 0 and less than or equal to 15;

[0010] When 1.5<the relative expression amount of Ve1 gene≤2.0, the disease index of the tested Solanum plant is greater than 15 and less than or equal to 35;

[0011] When 1.0<the relative expression amount of Ve1 gene≤1.5, the disease index of the tested Solanum plant is greater than 35 and less than or equal to 55;

[0012] When 0.1<the relative expression amount of Ve1 gene≤1.0, the disease index of the tested Solanum plant is greater than 55 and less than or equal to 75;

[0013] When the relative expression level of the Ve1 gene is ≤0.1, the disease index of the tested Solanum plant is greater than 75 and less than or equal to 100.

[0014] Preferably, the Verticillium wilt resistance of the tested Solanum plants is divided into high resistance, disease resistance, medium resistance, disease sensitivity and high sensitivity from high to low according to the disease index;

[0015] When 0<disease index≤15, the resistance of the tested Solanum plant to Verticillium wilt is high;

[0016] When 15<disease index≤35, the Verticillium wilt resistance of the tested Solanum plant is resistant;

[0017] When 35<disease index≤55, the resistance of the tested Solanum plant to Verticillium wilt is moderate;

[0018] When 55<disease index≤75, the Verticillium wilt resistance of the tested Solanum plant is susceptible;

[0019] When 75<disease index≤100, the resistance of the tested Solanum plant to Verticillium wilt is highly susceptible.

[0020] Preferably, the method for detecting the relative expression level of the Ve1 gene includes fluorescent quantitative PCR.

[0021] Preferably, the internal reference gene of the fluorescent quantitative PCR is 18sRNA.

[0022] Preferably, the 18sRNA amplification primers include an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.4.

[0023] Preferably, the method further comprises: a test experiment; when the disease index result of the tested Solanum plant determined according to the relative expression amount of the Ve1 gene is inconsistent with the result of the test experiment, re-detecting the relative expression amount of the Ve1 gene in the tested material and conducting a test experiment; the steps of the test experiment include:

[0024] An oblique incision is formed on the stem of the Solanum plant to be tested, the stem with the incision is immersed in a suspension of spores of the Verticillium wilt pathogen, and the upper and lower stem incisions after the immersion are attached and fixed to obtain the Solanum plant to be tested inoculated with the Verticillium wilt pathogen;

[0025] Cultivating the Solanum plant to be tested inoculated with the Verticillium wilt pathogen, observing the disease condition of the Solanum plant to be tested inoculated with the Verticillium wilt pathogen from the 5th day, observing once every 3 to 5 days, for a total of 5 times, and calculating the disease index based on the disease condition observed in the 5th time;

[0026] The disease index is calculated according to the disease condition of the tested Solanum plants inoculated with the Verticillium wilt pathogen;

[0027] The grading standard of the disease is as follows: Grade 0: no disease; Grade 1: only the first true leaf turns yellow or curls; Grade 2: the part below the third true leaf shows yellowing and leaves fall off; Grade 3: only one newly unfolded true leaf appears healthy, and the plant has obvious leaf drop; Grade 4: all unfolded true leaves fall off, and only one flag leaf remains on the plant; Grade 5: the plant is dead;

[0028] The disease index=[∑(disease grade value×number of plants with corresponding disease grade value)÷(5×total number of plants investigated)]×100.

[0029] Preferably, the concentration of the Verticillium wilt pathogen spore suspension is 1×10 7 spores / mL.

[0030] Preferably, the Verticillium wilt pathogen includes Verticillium dahliae.

[0031] Preferably, the soaking time is 10 to 15 minutes.

[0032] Preferably, the Solanum plant to be tested is a plant in the 6-8 true leaf stage.

[0033] Beneficial effects:

[0034] The present invention can determine the disease index of the tested Solanum plant by detecting the relative expression amount of the key disease resistance gene Ve1 of Verticillium wilt in Solanum plant and performing analysis. Compared with the traditional artificial inoculation identification method, the present invention has the advantages of being fast and efficient, and reducing time and economic costs.

[0035] Furthermore, the present invention uses the stem grafting inoculation method to artificially inoculate the pathogen of Verticillium wilt on the tested Solanum plants. Compared with the traditional root immersion method and root wounding method of artificial inoculation, the operation steps and the disease observation cycle are simplified (the traditional method requires observation starting from the 7th day after inoculation, and observation once every 7 days, for 4 times), and has the advantages of being fast, efficient, and highly repeatable; compared with the traditional leaf inoculation method (inoculation of a single leaf), the stem grafting inoculation method can grade the disease by observing the overall leaf and stem conditions, and the experimental results are more scientific and reliable, and the operability and repeatability are stronger.

[0036] Furthermore, the present invention can verify the two parts of the results by performing correlation analysis between the fluorescent quantitative PCR technology and the Verticillium wilt disease statistics, thereby reducing the errors caused by manual operation and the growth environment to the experiment, and improving the reliability, scientificity and accuracy of the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0038] Figure 1 It is the classification standard for leaf diseases of Solanum plants;

[0039] Figure 2 It is a standard for grading stem diseases of Solanum plants. DETAILED DESCRIPTION

[0040] The present invention provides a method for identifying resistance to Verticillium wilt in Solanum plants, comprising the following steps:

[0041] Four true leaf stage leaves of the Solanum plant to be tested are used as the test material, and the relative expression amount of the Ve1 gene in the test material is detected using a first primer pair, wherein the first primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2;

[0042] Determining the disease index of the Solanum plant to be tested according to the relative expression amount of the Ve1 gene in the tested material; when the relative expression amount of the Ve1 gene is greater than 2.0, the disease index of the Solanum plant to be tested is greater than 0 and less than or equal to 15;

[0043] When 1.5<the relative expression amount of Ve1 gene≤2.0, the disease index of the tested Solanum plant is greater than 15 and less than or equal to 35;

[0044] When 1.0<the relative expression amount of Ve1 gene≤1.5, the disease index of the tested Solanum plant is greater than 35 and less than or equal to 55;

[0045] When 0.1<the relative expression amount of Ve1 gene≤1.0, the disease index of the tested Solanum plant is greater than 55 and less than or equal to 75;

[0046] When the relative expression level of the Ve1 gene is ≤0.1, the disease index of the tested Solanum plant is greater than 75 and less than or equal to 100.

[0047] In the present invention, the Verticillium wilt disease classification standard, calculation method and disease resistance type classification are preferably based on the reports of Xiao Yunhua et al. [Xiao Yunhua, Lin Boqing. Identification of Verticillium wilt resistance of eggplant germplasm resources [J]. Chinese Vegetables, 1995 (1): 32-33.] and Li Xixiang et al. [Li Xixiang, Zhu Dewei. Eggplant germplasm resource description specification and data standard [M]. Beijing: China Agricultural Press, 2006.].

[0048] In the present invention, the Verticillium wilt resistance of the tested Solanum plant is preferably divided into high resistance, disease resistance, medium resistance, disease sensitivity and high sensitivity from high to low according to the disease index; the specific preferred methods include:

[0049] When 0<disease index≤15, the resistance of the tested Solanum plant to Verticillium wilt is high;

[0050] When 15<disease index≤35, the Verticillium wilt resistance of the tested Solanum plant is resistant;

[0051] When 35<disease index≤55, the resistance of the tested Solanum plant to Verticillium wilt is moderate;

[0052] When 55<disease index≤75, the Verticillium wilt resistance of the tested Solanum plant is susceptible;

[0053] When 75<disease index≤100, the resistance of the tested Solanum plant to Verticillium wilt is highly susceptible.

[0054] In the present invention, the method for detecting the relative expression level of the Ve1 gene preferably includes fluorescent quantitative PCR; the internal reference gene of the fluorescent quantitative PCR is preferably 18sRNA; the amplification primers of the 18sRNA include an upstream primer whose nucleotide sequence is preferably shown in SEQ ID NO.3 and a downstream primer whose nucleotide sequence is shown in SEQ ID NO.4.

[0055] The present invention analyzes the relative expression of the key disease resistance gene Ve1 of eggplant Verticillium wilt by using fluorescent quantitative PCR technology, and can determine the disease index of the tested Solanum plant. Compared with the traditional artificial inoculation identification method, the method is fast and efficient, and reduces time and economic costs.

[0056] In the present invention, the method preferably also includes: a test experiment; when the disease index result of the Solanum plant to be tested determined according to the relative expression amount of the Ve1 gene is inconsistent with the result of the test experiment, re-detecting the relative expression amount of the Ve1 gene in the test material and conducting the test experiment until the two results are consistent, thereby reducing the error caused by manual operation and growth environment to the experiment, and improving the reliability, scientificity and accuracy of the experimental results.

[0057] The steps of the test experiment of the present invention preferably include:

[0058] When the Solanum plant to be tested grows to 6 to 8 true leaves, the stem of the Solanum plant to be tested is cut obliquely from the middle to form an oblique incision on the stem, the stem with the incision is disinfected and then immersed in a suspension of spores of the Verticillium wilt pathogen, the upper and lower stem incisions after immersion are attached and fixed, and the Solanum plant to be tested inoculated with the Verticillium wilt pathogen is obtained.

[0059] After the stem of the Solanum plant to be tested is cut obliquely from the middle, the stem incision with the root is preferably recorded as the lower incision, and the stem incision with the top is preferably recorded as the upper incision. In the present invention, the upper incision is preferably immersed in a suspension of spores of the Verticillium wilt pathogen; the concentration of the suspension of spores of the Verticillium wilt pathogen is preferably 1×10 7 spores / mL; the Verticillium wilt pathogen preferably includes Verticillium dahliae; the soaking time is preferably 10 to 15 minutes; after the soaking, the soaked stem is preferably rinsed with sterile water to avoid excessive bacterial liquid or spores on the plant surface, which is easy to cause environmental pollution. After the sterile water rinse, the upper incision and the lower incision after the soaking are preferably attached and fixed to obtain the Solanum plant to be tested inoculated with the Verticillium wilt pathogen.

[0060] After obtaining the Solanum plant to be tested inoculated with the Verticillium wilt pathogen, the present invention preferably cultivates the Solanum plant to be tested inoculated with the Verticillium wilt pathogen, observes the disease condition of the Solanum plant to be tested inoculated with the Verticillium wilt pathogen from the 5th day, observes once every 3 to 5 days, and observes 5 times in total, and calculates the disease index based on the disease condition of the last observation (i.e., the 5th time) (i.e., the disease index is calculated based on the disease condition of the last observation and statistics); calculates the disease index based on the disease condition of the Solanum plant to be tested inoculated with the Verticillium wilt pathogen;

[0061] The grading standard of the disease is as follows: Grade 0: no disease; Grade 1: only the first true leaf turns yellow or curls; Grade 2: the part below the third true leaf shows yellowing and leaves fall off; Grade 3: only one newly unfolded true leaf appears healthy, and the plant has obvious leaf drop; Grade 4: all unfolded true leaves fall off, and only one flag leaf remains on the plant; Grade 5: the plant is dead;

[0062] The disease index=[∑(disease grade value×number of plants with corresponding disease grade value)÷(5×total number of plants investigated)]×100.

[0063] The present invention can avoid accidental events by observing the disease conditions of plants for five consecutive times, thereby improving the reliability and scientificity of the experimental results.

[0064] In the present invention, the day and night temperatures of the culture are preferably 25° C. and 16° C., respectively, and the humidity of the culture is preferably 70% to 80%.

[0065] The present invention uses a stem grafting inoculation method to artificially inoculate the Solanum material to be identified with Verticillium wilt. Compared with a traditional root immersion method and a root injury method of artificial inoculation, the present invention simplifies the operation steps and the disease observation cycle (the traditional method requires observation starting from the 7th day after inoculation, and observation once every 7 days, for 4 times), and has the advantages of being fast, efficient and highly repeatable. Compared with a traditional leaf inoculation method (inoculation of a single leaf), the stem grafting inoculation method can grade the disease by observing the overall leaf and stem conditions, and the experimental results are more scientific and reliable, and the operability and repeatability are stronger.

[0066] The present invention conducts correlation analysis between fluorescent quantitative PCR technology and Verticillium wilt disease statistics, and can verify the two parts of the results with each other, thereby reducing the errors caused by manual operation and growth environment to the experiment, improving the reliability, scientificity and accuracy of the experimental results, and can verify and accurately identify the Verticillium wilt resistance of Solanum materials to be identified.

[0067] In order to further illustrate the present invention, a method for identifying resistance to Verticillium wilt of Solanum provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] 1. Use fluorescent quantitative PCR (qRT-PCR) to analyze the expression of target genes

[0070] (1) Primer design

[0071] According to the cDNA sequence of Ve1 in the reference material Solanum sisymbriifolium Lam. (gene accession number: BankIt2448495), the BLAST software of NCBI was used to search for the reported Ve1 homologous genes in Solanaceae, namely StVe1 in S. torvum, SaVe1 in S. aethiopicum, SlVe1 in S. lycopersicum and Ve1 in S. tuberosum; DNAMAN software was used to analyze the sequence of the above Solanaceae Ve1 homologous genes, find the gene fragments with consistent sequences, and design the corresponding qRT-PCR primers. The internal reference gene is 18sRNA, and the primer sequences are as follows:

[0072] Ve1-F: 5'-ATGAGATTCTTACACCTTCTATGGC-3', SEQ ID NO.1;

[0073] Ve1-R: 5'-TTTGCTGCCCCTACTATGTATCC-3', SEQ ID NO.2;

[0074] 18sRNA-F: 5'-TAGTTGGACTTTGGGATGGC-3', SEQ ID NO.3;

[0075] 18sRNA-R: 5'-AGAGCGTAGGCTTGCTTTGA-3', SEQ ID NO.4;

[0076] (2) Template preparation

[0077] Extract RNA from leaves of materials, apply 0.8% agarose gel electrophoresis at 150V for 20min, and after passing the integrity test, use reverse transcription kit to reverse transcribe RNA into cDNA;

[0078] (3) Gene expression analysis

[0079] The cDNA in step (2) was diluted twice and used as a template. The primers in step (1) were used to carry out the qRT-PCR test. The specific qRT-PCR reaction procedure was carried out according to the instructions of the kit (2×TSINGKE Master qPCR Mix (SYBR Green I) Fluorescence Quantitative PCR Kit produced by Beijing Qingke Xinye Biotechnology Co., Ltd.). 2 −△△CT Methods The relative expression of genes was calculated.

[0080] 2. Use the artificial stem grafting inoculation method of Verticillium wilt to analyze the disease of the material

[0081] (1) Preparation of spore solution

[0082] The pathogen of Verticillium wilt used in this example is Verticillium dahliae QZ-S, which is preserved in the Eggplant Breeding Laboratory of the Institute of Horticultural Crops, Yunnan Academy of Agricultural Sciences and published in the literature [Wu Liyan, Guo Zhixiang, Zeng Li, et al. Identification and analysis of resistance to Verticillium wilt in Yunnan wild eggplant resources at the seedling stage by artificial inoculation [J]. Journal of Plant Genetic Resources, 2017, 18(6):9. DOI:10.13430 / j.cnki.jpgr.2017.06.007.]. 500 µL of the Verticillium wilt pathogen (containing 50% glycerol) stored at -80°C was taken into a 2mL centrifuge tube, and 500 µL of PS culture medium (potato liquid culture medium) was added, and the culture was activated on a shaker at 28-30°C and 200 r / min for 2-3 hours; the activated bacterial solution was poured into a 500mL Erlenmeyer flask containing 100mL PS culture medium (containing ampicillin at a working concentration of 75 mg / L), and cultured on a shaker at 25-28°C and 180 r / min for 5-7 days; the bacterial solution was filtered with double gauze, the filtrate was retained, and the filtrate was centrifuged at 1500-2000 r / min at room temperature for 10-15 minutes, the supernatant was discarded, and the spore precipitate was retained; the spore precipitate was diluted with sterile water, and counted with a 16×25 hemocytometer, and the bacterial solution was configured to a concentration of 1×10 7 of spore suspension.

[0083] (2) Inoculation

[0084] When the plant grows to 6-8 true leaves, use a sterilized single-sided blade to cut the stem of the plant diagonally from the middle, and soak the lower end of the stem with the top in 2% sodium hypochlorite for 3-5 minutes; then sterilize it in a concentration of 1×10 7 Soak the plants in the spore suspension for 10-15 minutes, take them out and rinse them with sterile water for 3 times, then wrap and fix the cuts of the upper and lower stems with plastic wrap, or use grafting tubes or grafting clips to fix them. The plants are cultured in an incubator at a day and night temperature of 25℃ / 16℃ and a humidity of 70%-80%. The first investigation begins on the 5th day after treatment, and observations are made every 3 days for 4 consecutive times, for a total of 5 times. The disease index is calculated based on the last observation and statistics of the disease situation.

[0085] (3) Disease statistics

[0086] The classification standard, calculation method and classification of disease resistance types of Verticillium wilt disease refer to the reports of Xiao Yunhua et al. [Xiao Yunhua, Lin Boqing. Identification of Verticillium wilt resistance of eggplant germplasm resources [J]. Chinese Vegetables, 1995 (1): 32-33.] and Li Xixiang et al. [Li Xixiang, Zhu Dewei. Eggplant germplasm resource description specification and data standard [M]. Beijing: China Agricultural Press, 2006.] Disease classification standard: Grade 0: disease-free; Grade 1: only the first true leaf turns yellow or curls; Grade 2: the part below the third true leaf shows yellowing and leaves fall off; Grade 3: only one newly expanded true leaf appears healthy, and the plant has obvious leaf drop; Grade 4: all expanded true leaves fall off, and only one flag leaf remains on the plant; Grade 5: plant death.

[0087] Calculate the disease rate and disease index of each strain and each level. For specific plant disease grading standards, please refer to Figure 1 (leaves) and Figure 2 (stem), disease index (DI) = [∑ (grade value × number of plants with corresponding grade value) ÷ (5 × total number of plants investigated)] × 100. The population resistance of germplasm resources is divided into 6 levels according to the disease index at the seedling stage, namely immune (disease index = 0, no infection), highly resistant (0 < disease index ≤ 15), resistant (15 < disease index ≤ 35), moderately resistant (35 < disease index ≤ 55), susceptible (55 < disease index ≤ 75) and highly susceptible (75 < disease index ≤ 100).

[0088] 3. Joint analysis of disease resistance gene expression and disease index

[0089] (1) The bivariate-Pearson correlation analysis method in SPSS software was used to perform correlation analysis between the Verticillium wilt disease index of the material in step 2 and the relative expression of the Ve1 gene in step 1. The relative coefficient between the disease index of the material and the relative expression of the Ve1 gene is R, where a positive R value represents a positive correlation between the two, and a negative R value represents a negative correlation between the two; P<0.01 represents that the difference between different materials is at an extremely significant level, and P<0.05 represents that the difference is at a significant level.

[0090] (2) Through the correlation analysis (Table 2) of 30 eggplant materials with different resistance (Table 1), it can be seen that the disease index of the material is extremely significantly negatively correlated with the relative expression of the Ve1 gene (P value is 0.006, correlation coefficient R is -0.479), that is, the higher the relative expression of the Ve1 gene, the better the material's resistance to Verticillium wilt and the milder the disease. The relative expression of the Ve1 gene can represent the material's resistance to Verticillium wilt to a certain extent.

[0091] Table 1 Sources of eggplant materials tested

[0092] Resource Number Resource Name source Resource Number Resource Name source 197 Garlic Mustard Eggplant Kunming, Yunnan 249-1 Solanum simaoensis Pu'er City, Yunnan 2-1 Cassia Xishuangbanna, Yunnan 245-4-2 Solanum simaoensis Dali City, Yunnan 250 Water Eggplant Pu'er City, Yunnan 121-3 Mengla Solanum Mengla City, Yunnan 180 Solanum spicata Pu'er City, Yunnan 167 Solanum simaoensis Pu'er City, Yunnan 194 Red tomato Jianshui City, Yunnan 246-3-1 Solanum simaoensis Pu'er City, Yunnan 193-1 Red tomato Lancang City, Yunnan 249-3-2 Solanum simaoensis Pu'er City, Yunnan 367 Beef Eggplant Lancang City, Yunnan 246-3-2 Solanum simaoensis Pu'er City, Yunnan 192 Red tomato Dali City, Yunnan 247-2 Solanum simaoensis Pu'er City, Yunnan 105 Tengchong Red Eggplant Tengchong City, Yunnan 248-2-1 Solanum simaoensis Pu'er City, Yunnan 243 Solanum multifiss Pu'er City, Yunnan 246-4-2 Solanum simaoensis Pu'er City, Yunnan 240-4-3 Dali wild eggplant Dali City, Yunnan 143 Jinghong Solanum Jinghong City, Yunnan 241-2-2 Dali wild eggplant Dali City, Yunnan 242 Kunming wild eggplant Kunming, Yunnan 107 Solanum sibannaense Xishuangbanna, Yunnan 239-3-2 Dali wild eggplant Dali City, Yunnan 141-1 Dali wild eggplant Dali City, Yunnan 245-1-1 Solanum simaoensis Pu'er City, Yunnan 249-4-1 Solanum simaoensis Pu'er City, Yunnan 251-5-1 Solanum simaoensis Pu'er City, Yunnan

[0093] Table 2 Correlation analysis between disease index and Ve1 gene expression in different eggplant materials

[0094] Serial number Resource Number Disease index Relative expression of Ve1 gene Serial number Resource Number Disease index Relative expression of Ve1 gene 1 197 (control) 8.00 2.46 17 249-3-2 73.30 0.17 2 2-1 12.70 2.03 18 246-4-2 73.30 0.11 3 250 15.20 1.80 19 247-2 74.00 0.12 4 243 28.00 1.53 20 246-3-2 74.70 0.15 5 180 43.30 1.00 21 240-4-3 76.00 0.04 6 193-1 46.00 1.08 22 241-2-2 76.70 0.06 7 367 47.20 1.00 23 107 78.00 0.00 8 194 49.30 1.19 24 141-1 78.00 0.00 9 192 49.30 1.09 25 249-4-1 80.70 0.06 10 105 51.30 1.16 26 143 81.30 0.00 11 249-1 55.30 0.51 27 245-1-1 82.70 0.01 12 245-4-2 58.70 0.57 28 251-5-1 82.70 0.01 13 121-3 61.30 0.11 29 242 86.00 0.05 14 167 64.70 0.16 30 239-3-2 88.00 0.01 15 246-3-1 65.30 0.14 / R-Value / -0.479** 16 248-2-1 71.30 0.22 / P-value / 0.006

[0095] (3) From Table 2, we can see that the relative expression of Ve1 gene in highly resistant materials No. 1-2 (0<disease index≤15) is higher than 2.0, the relative expression of Ve1 gene in resistant materials No. 3-4 (15<disease index≤35) is between 1.5 and 2.0, the relative expression of Ve1 gene in medium-resistant materials No. 5-10 (35<disease index≤55) is generally around 1.0, the relative expression of Ve1 gene in susceptible materials No. 11-20 (55<disease index≤75) is generally between 0.1 and 1.0, and the relative expression of Ve1 gene in highly susceptible materials No. 21-30 (75<disease index≤100) is generally lower than 0.1. Therefore, the relative expression of Ve1 gene in the leaves of materials can be used to quickly divide the materials into the Verticillium wilt resistance, as shown in Table 3.

[0096] Table 3 Corresponding relationship between relative expression of Ve1 gene and resistance to Verticillium wilt

[0097] Relative expression of Ve1 gene X＞2.0 1.5＜X≤2.0 1.0＜X≤1.5 0.1＜X≤1.0 X≤0.1 Verticillium wilt resistance High resistance Disease resistance Zhongkang Disease High Sensitivity

[0098] Note: X in Table 3 is the relative expression level of Ve1 gene in leaves of materials.

[0099] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for identifying resistance to Verticillium wilt in Solanum, characterized in that: The following steps are involved: Four true leaf stage leaves of the Solanum plant to be tested are used as the test material, and the relative expression amount of the Ve1 gene in the test material is detected using a first primer pair, wherein the first primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; Determining the disease index of the Solanum plant to be tested according to the relative expression amount of the Ve1 gene in the tested material; when the relative expression amount of the Ve1 gene is greater than 2.0, the disease index of the Solanum plant to be tested is greater than 0 and less than or equal to 15; When 1.5<the relative expression amount of Ve1 gene≤2.0, the disease index of the tested Solanum plant is greater than 15 and less than or equal to 35; When 1.0<the relative expression amount of Ve1 gene≤1.5, the disease index of the tested Solanum plant is greater than 35 and less than or equal to 55; When 0.1<the relative expression amount of Ve1 gene≤1.0, the disease index of the tested Solanum plant is greater than 55 and less than or equal to 75; When the relative expression level of the Ve1 gene is ≤0.1, the disease index of the tested Solanum plant is greater than 75 and less than or equal to 100.

2. The method according to claim 1, characterized in that According to the disease index, the Verticillium wilt resistance of the tested Solanum plants is divided into high resistance, disease resistance, medium resistance, disease sensitivity and high sensitivity from high to low; When 0<disease index≤15, the resistance of the tested Solanum plant to Verticillium wilt is high; When 15<disease index≤35, the Verticillium wilt resistance of the tested Solanum plant is resistant; When 35<disease index≤55, the resistance of the tested Solanum plant to Verticillium wilt is moderate; When 55<disease index≤75, the Verticillium wilt resistance of the tested Solanum plant is susceptible; When 75<disease index≤100, the resistance of the tested Solanum plant to Verticillium wilt is highly susceptible.

3. The method according to claim 1, characterized in that The method for detecting the relative expression level of the Ve1 gene includes fluorescence quantitative PCR.

4. The method according to claim 3, characterized in that The internal reference gene of the fluorescent quantitative PCR is 18sRNA.

5. The method according to claim 4, characterized in that The 18sRNA amplification primers include an upstream primer whose nucleotide sequence is shown as SEQ ID NO.3 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.

4.

6. The method according to claim 1, characterized in that The method further comprises: a test experiment; when the disease index result of the tested Solanum plant determined according to the relative expression amount of the Ve1 gene is inconsistent with the result of the test experiment, re-detecting the relative expression amount of the Ve1 gene in the tested material and conducting a test experiment; the steps of the test experiment include: An oblique incision is formed on the stem of the Solanum plant to be tested, the stem with the incision is immersed in a suspension of spores of the Verticillium wilt pathogen, and the upper and lower stem incisions after the immersion are attached and fixed to obtain the Solanum plant to be tested inoculated with the Verticillium wilt pathogen; Cultivating the Solanum plant to be tested inoculated with the Verticillium wilt pathogen, observing the disease condition of the Solanum plant to be tested inoculated with the Verticillium wilt pathogen from the 5th day, observing once every 3 to 5 days, for a total of 5 times, and calculating the disease index based on the disease condition observed in the 5th time; The disease index is calculated according to the disease condition of the tested Solanum plants inoculated with the Verticillium wilt pathogen; The grading standard of the disease is as follows: Grade 0: no disease; Grade 1: only the first true leaf turns yellow or curls; Grade 2: the part below the third true leaf shows yellowing and leaves fall off; Grade 3: only one newly unfolded true leaf appears healthy, and the plant has obvious leaf drop; Grade 4: all unfolded true leaves fall off, and only one flag leaf remains on the plant; Grade 5: the plant is dead; The disease index=[∑(disease grade value×number of plants with corresponding disease grade value)÷(5×total number of plants investigated)]×100.

7. The method according to claim 6, characterized in that The concentration of the Verticillium wilt pathogen spore suspension is 1×10 7 spores / mL.

8. The method according to claim 6 or 7, characterized in that: The Verticillium wilt pathogen includes Verticillium dahliae.

9. The method according to claim 6, characterized in that The soaking time is 10 to 15 minutes.

10. The method according to claim 6, characterized in that The Solanum plant to be tested is a plant in the stage of 6 to 8 true leaves.

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