A method for evaluating the transport capacity of wheat ear tissue for DON toxin and its application

Abstract

The present invention discloses a method for evaluating the transport capacity of wheat spike tissue for DON toxin and its application. The present invention mainly detects the absolute value of DON toxin content in different parts of wheat spike inoculated with Gibberella and calculates the transport capacity of wheat spike tissue for DON toxin. Using this phenotypic evaluation method, combined with whole genome association analysis, the inventor identified a site significantly associated with DON toxin transport capacity, located at 463,808,261bp-497,210,220bp interval of chromosome 1A. The present invention can not only be used for the screening and creation of low-toxin wheat germplasm, but also can be used to dig out QTL or genes related to wheat toxin transport capacity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a phenotypic identification and evaluation method related to fusarium fusarium, and specifically relates to a method for evaluating the transport capacity of wheat ear tissue to DON toxin produced by fusarium fusarium, and its application in screening low DON toxin germplasm and localization of transport-related genes. Background Art

[0002] Fusarium head blight (FHB) is a disease caused by Fusarium graminearum complex, which not only causes serious loss of wheat yield, but also enriches DON toxins produced by Fusarium graminearum in the grains during the disease process. After the occurrence of fusarium head blight in the wheat ear, the grains infected by Fusarium graminearum develop abnormally and cannot be filled normally, resulting in shriveled and deformed seeds. The parts not infected by Fusarium graminearum can form plump grains, but the DON toxins in these seemingly normal grains are seriously excessive. The deformed grains are easy to identify and remove in the later processing, and the accumulated DON toxins will not enter the food chain. On the contrary, the grains with normal appearance but excessive DON toxins are more likely to be ignored, resulting in excessive starch DON toxins after processing, threatening food safety. Therefore, controlling the DON toxins in normal grains on diseased wheat ears is the key to controlling toxins in food, but there is still no economical and environmentally friendly way to control toxins in grains. According to research findings, the DON toxin in normal grains may originate from diseased tissues, but the genetic mechanism behind how the DON toxin is transported from diseased tissues to normal grains remains unclear. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a more accurate and reasonable method for phenotypic identification and evaluation of the DON toxin transport capacity of wheat ear tissue. By correlating this transport capacity with the genotype of different varieties of wheat, the DON toxin transport-related genes can be located and cloned, and can also be used to screen wheat varieties with low toxin accumulation capacity.

[0004] Technical solution: In order to solve the above technical problems, the present invention provides a method for evaluating the transport capacity of wheat ear tissue for DON toxin, comprising the following steps:

[0005] 1) During the flowering stage of wheat ears, two florets of any spikelet from the 1st to the 6th spikelet from the top of the wheat ear were inoculated with spores of Fusarium graminearum;

[0006] 2) According to the severity of the disease, the ears are divided into infected spikelets, transitional spikelets and non-infected spikelets; the infected spikelets are dry, no plump seeds are formed, the spike axis is browned and / or severely browned, and if the air humidity reaches more than 70%, this part will be covered with white hyphae; the seeds of the transitional spikelets are full, the husk is white, and the spike axis is not browned; the non-infected spikelets are no different from normal spikelets, and the grains are full;

[0007] 3) The toxin content of the spikelets in the infected area is named DON IS The toxin content in the spikelets of the non-infected area is named DON US , the calculation formula for toxin transport capacity is established as:

[0008]

[0009] 4) When DTC≤0.2, the wheat variety is considered to have low DON transport capacity; when DTC≥0.7, the wheat variety is considered to have high DON transport capacity.

[0010] Wherein, the concentration of the spore solution of Fusarium graminearum in step 1) is 1×10 5 / mL, and each spikelet was inoculated with 10μL.

[0011] The method for detecting the toxin content in the spikelets in the infected area in step 3) includes: taking spike tissues of multiple independent wheat ears in the infected area and mixing them as a sample, grinding them into powder, and then detecting the toxin by liquid chromatography-mass spectrometry to obtain DON. IS .

[0012] In step 3), the ear tissues of multiple independent wheat ears in the non-infected area are mixed as a sample, ground into powder, and then the toxin is detected by liquid chromatography-mass spectrometry to obtain DON. US .

[0013] The present invention also includes the use of the method in gene localization or in screening wheat varieties with low toxin or low transport capacity.

[0014] Wherein, the low-toxin wheat variety is a wheat variety with DTC≤0.2.

[0015] Among them, the wheat varieties with low toxin or low transport capacity include Xindong 20, Shimai 19, Qingchun 5, Jining 13, Kaimai 21, and Keyi 26.

[0016] The present invention also includes a method for screening low-toxin wheat varieties or low-transport capacity wheat varieties, comprising the following steps: using the method to screen wheat varieties with DTC≤0.2.

[0017] Beneficial effects: The present invention has for the first time obtained an effective and stable method for analyzing and evaluating the DON toxin transport capacity of wheat ear tissue, which can be used for the screening and creation of low-DTC wheat germplasm and the positioning and cloning of DTC-related genes, which is of great significance and application prospects for ensuring the quality and safety of grain. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , Flow chart of ear sampling and DON detection after Fusarium graminearum inoculation. A: Inoculation of Fusarium graminearum by double flower drip method. The sixth spikelet pointed by the syringe is the inoculated spikelet, and the blue dot represents the inoculated floret. B: Phenotype of successfully inoculated spikelets at maturity. According to the degree of infection of Fusarium graminearum, it is divided into three parts. The upper part is the spikelet (IS) completely infected by Fusarium graminearum, with no grain formation or shriveled grains; the middle part is the part caused by the downward expansion of Fusarium graminearum from the upper part, and the husk of the spikelet shows the classic bleaching of Fusarium graminearum; the lower part is the non-infected area (US), with no symptoms of Fusarium graminearum, but full grains. C: The IS area of ​​three independent ears was collected, mixed, ground, and the toxin content in this area was detected by liquid chromatography-mass spectrometry. D: The US area of ​​three independent ears was collected, mixed, ground, and the toxin content in this area was detected by liquid chromatography-mass spectrometry.

[0019] Figure 2 Frequency histogram of the number of diseased ears in XP and XSB populations.

[0020] Figure 3 , DTC histogram of XP and XSB populations detected by ear separation method.

[0021] Figure 4 , The gene localization results of DTC in XP and XSB populations were analyzed by ear division method.

[0022] Figure 5 , DON standard curve. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below in conjunction with examples. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0024] A genome-wide association study (GWAS) of wheat DTC was conducted using a natural wheat population to obtain QTL loci closely linked to DTC. The specific implementation method is as follows:

[0025] 1. Material selection

[0026] In order to evaluate the effectiveness of this method, we selected two groups composed of different wheat varieties for testing, named XP group and XSB group respectively. The overall resistance of the groups is different. Most of the XP group are disease-resistant varieties, mostly domestic varieties, and varieties from the middle and lower reaches of the Yangtze River account for 60%; the resistance to ergot disease in the XSB group is mostly moderate, and the resistance follows a normal distribution. They come from many regions of the world and are widely representative. The number of varieties in the XP group and the XSB group are 125 and 130 respectively. The details of the group varieties are shown in Tables 1 and 2. Among them, the average number of diseased ears in the XP group is about 6.5, and the average number of diseased ears in the XSB group is about 9.5. In comparison, most varieties in the XP group are relatively resistant to ergot disease. Figure 2 A shows that nearly 40% of the varieties in this group are highly resistant varieties. The resistance to fusarium head blight in the XSB group shows a normal distribution, with most of the materials being moderately susceptible ( Figure 2 B).

[0027] Table 1. Varieties and origins of XP populations

[0028]

[0029]

[0030]

[0031] The selection of XP group samples covers four countries including the United States, South Korea, Japan and China, among which the varieties from Jiangsu, China account for the highest proportion.

[0032] Table 2. Varieties and origins of the xsB population

[0033]

[0034]

[0035] The sample selection of the XSB group involved 14 regions, among which varieties from the United States and China accounted for the largest proportion, and in my country, the largest number of varieties were selected from Shandong Province.

[0036] 2. Conduct genotyping analysis on the materials of the two groups.

[0037] The XP genotype was analyzed using wheat 55K SNP chip, and the XSB population was genotyped by transcriptome sequencing.

[0038] 2.1. DNA extraction from XP population materials

[0039] (1) Take about 3 cm of wheat leaves from each variety and place them in a 2 mL centrifuge tube;

[0040] (2) Add one grinding steel ball, cover the lid tightly, place it in liquid nitrogen for 60 seconds, and then grind the sample using a grinding machine for 30 seconds at a grinding frequency of 1400 r / min;

[0041] (3) Add 1000 μl of 1.5% CTAB extract to the ground sample, place in a 70°C oven, let stand for 1 h, and mix by inverting every 20 min;

[0042] (4) Cool to room temperature, add 500 μL of chloroform, mix by inversion, and centrifuge at 12000 r / min for 5 min;

[0043] (5) Pipette 600 μL of the supernatant into a new 1.5 ml centrifuge tube, add an equal amount of isopropanol, mix by inverting 10 times, let stand at -20°C for 1 h; centrifuge at 12000 r / min for 5 min, and discard the supernatant;

[0044] (6) Add 1 mL of 75% alcohol for cleaning;

[0045] (7) Balanced centrifugation at 12000 r / min for 5 min, discard the alcohol, absorb the excess liquid, and blow dry at room temperature;

[0046] (8) Add 50 μL of ddH2O and dissolve at room temperature for 30 min. Store at -20°C for later use or send to Beijing Zhongyu Gold Labeling (Beijing) Biotechnology Co., Ltd. for chip analysis.

[0047] 2.2. Extraction of RNA from XSB population materials

[0048] The mortar was treated at 180℃ for 8h or burned to eliminate RNA enzyme contamination; chloroform, isopropanol, ethanol and other reagents were newly opened and uncontaminated; other equipment such as gun tips, centrifuge tubes and reagents such as ultrapure water and NaAc were treated with 1‰ DEPC water overnight and then sterilized at 121℃ for 30min; gun tips and centrifuge tubes were dried at 65℃ for later use; Trizol method was used to extract mRNA of each wheat variety in XSB population:

[0049] (1) Grind about 100 mg of wheat leaves with liquid nitrogen, add 1 mL of RNAiso Plus reagent (Takara, Code No.: 9109), and after thorough homogenization, transfer the homogenate into a 1.5 mL centrifuge tube and place at room temperature for 5 min.

[0050] (2) Add 200 μl of chloroform, shake and mix, let stand for 5 min, and centrifuge at 4°C, 12,000 rpm for 10 min;

[0051] (3) Take the supernatant and transfer it to another centrifuge tube. Add an equal volume of isopropanol, shake and mix, precipitate at -20°C for 30 min, and centrifuge at 4°C, 12,000 rpm for 10 min.

[0052] (4) Discard the supernatant, wash with 1 mL of 75% ethanol (prepared with DEPC-treated sterile water), centrifuge at 4°C, 12,000 r / min for 10 min, air-dry at room temperature for about 10 min, add 40 μL of DEPC-treated RNase Free water (DEPC water) to dissolve the precipitate, store at -80°C for later use or directly send to BGI for transcriptome sequencing to analyze the SNP information of the variety.

[0053] 3. Phenotypic identification.

[0054] 3.1 Preparation of Fusarium graminearum spore solution

[0055] The strain used in this experiment was the sequenced Fusarium graminearum strain PH-1. It was inoculated in PDA medium and placed in a 25°C incubator for 5 days. Then, holes were punched on the grown bacterial plate and placed in a mung bean soup liquid medium at 25°C, 150r / min, and shaken for 3-5 days. 10μL of spores were dropped on a blood cell counting plate and then observed under a microscope. When the concentration reached 1×10 5 pcs / mL can be used for inoculation.

[0056] 3.2 Inoculation

[0057] During the wheat flowering period, Figure 1 The double-flower drip method shown in A was used to inoculate, and 10 μL of the prepared Gibberella spore solution (concentration 1×10 5 / mL), inoculated in the middle of the inner and outer lemma of the florets on both sides of the sixth spikelet, marked on the rachis of the inoculated spikelet and recorded the inoculation time, and the number of inoculated spikelets was more than 7 independent spikes.

[0058] 3.3 Phenotypic identification and sampling

[0059] On the 21st day after inoculation, the number of diseased spikelets on each ear of each variety was counted and group resistance and susceptibility analysis was performed. The results were as follows: Figure 2 On the 25th day after inoculation, the grains were full, the disease was stable, the ears were yellow, and basically mature. Figure 1 As shown in C and 1D, diseased and non-diseased ears were collected, and 3 independent replicates were taken and mixed as a sample, which was put into a kraft paper bag and placed in an oven to dry at 80°C.

[0060] 3.4 Grinding

[0061] After all samples are collected and dried, they are ground using a grinding machine. The parameters are set as follows: frequency = 1200 times / minute, time 5 minutes. After grinding, weigh and obtain the total weight data (see Table 3 and Table 4), then take out 0.5g and put it into a 10mL centrifuge tube for toxin detection. After the data is detected, the absolute content of DON in the sample is calculated based on the total weight.

[0062] 3.5 Toxin pretreatment

[0063] (1) Preparation of extraction solution and dissolution solution (carried out in a fume hood)

[0064] Extraction solution: 49.5% acetonitrile (analytical grade) + 1% formic acid (analytical grade) + 49.5% ddH2O

[0065] Dissolving solution: 49.5% acetonitrile (chromatographic grade) + 1% formic acid (chromatographic grade) + 49.5% ddH2O

[0066] (2) Add 4 mL of the extract to a 10 mL centrifuge tube containing 0.5 g of wheat flour and shake rapidly;

[0067] (3) Place in a shaking incubator at an angle of 160 rpm for 30 min;

[0068] (4) Centrifugation, 3400 r / min, 5 min;

[0069] (5) Prepare another 10 ml tube, write the number, add 500 mg of purification salt package (CNW technologies, Cat: 1059384, Lot: 55870030) to each tube, draw 2 ml of supernatant into each tube, and shake rapidly for 1 min;

[0070] (6) Centrifugation, 3400 r / min, 5 min;

[0071] (7) Write the number on the glass tube and aspirate 300 μL of supernatant to the bottom of the tube;

[0072] (8) Nitrogen blowing;

[0073] (9) Add 500 μl of dissolving solution to the bottom of the tube and shake gently to dissolve;

[0074] (10) Insert a disposable syringe, suck up all the solution, remove the needle, and replace the filter membrane;

[0075] (11) Slowly filter into the inner tube of the small brown bottle

[0076] (12) Store at -20°C and wait for testing.

[0077] 3.6 Detection of DON toxin by LC-MS

[0078] This example uses a triple quadrupole liquid-mass spectrometer (TSQ-Vantage, Thermo Fisher SCIENTIFIC). ) Chromatographic conditions: The chromatographic column is ACQUITY UPLC HSS T3 (2.1*100mm, 1.8um); the injection volume is 10ul; the flow rate is 0.35ml / min; the column temperature is 40°C; and the mobile phase consisting of an aqueous phase and an organic phase (B) is used for gradient elution. Elution program: 0-0.5min, 90%-65% (A), 10%-35% (B); 0.5-1 min, 65%-50% (A), 35%-50% (B); 1.5-5min, 35%-10% (A), 65%-90% (B); 5-8min, 10%-90% (A), 90%-10% (B); Mass spectrometry conditions: Use positive ion mode, DON peak time is generally 2.88 minutes; First, measure DON gradient standard sample on the machine, and obtain the DON determination standard curve (see Figure 5 ), the DON content in the sample was calculated according to the standard curve after the sample test results, and the absolute content of DON in each sample was calculated according to the test amount and the total mass of the sample (see Table 3 and Table 4).

[0079] Table 3 XP group toxin detection data

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Table 4 XSB group toxin detection data

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] 3.7 Calculation of Toxin Transport Capacity (DTC)

[0093] Based on toxin test results, it is known that DON US and DON IS After the data is collected, follow the formula: The results of DTC calculation (see Table 3 and Table 4) and data analysis of DTC were performed. According to the embodiment adopted by the present invention, it can be found that in the XP and XSB groups, the data of DTC showed a normal distribution ( Figure 3 ), indicating that DTC also exhibits the characteristics of quantitative traits. 3.8 Genome-wide association analysis of DTC

[0094] In the present embodiment, the mixed linear model (MLM) in the R package GAPIT3 was used to perform genome-wide association analysis on the DTC phenotype data sets of the two groups ( Figure 4 ), the marker with significant level (-log10(P)>3) was considered as a valid site, and the Manhattan plot was drawn using R package CMplot ( Figure 4 ) and visualized the results. The results showed that in the two different varieties of populations, there was a repeatable site that was significantly associated with DTC, located in the long arm of chromosome 1A, 463,808,261bp-497,210,220bp interval, indicating that the method is relatively stable.

[0095] 3.9 Screening of low DTC wheat varieties

[0096] Using the method of the present invention, 6 wheat varieties with DTC≤0.2 were screened (Table 5).

[0097] Table 5 List of wheat germplasms with low DTC screened using the method of the present invention

[0098]

[0099] These wheat varieties are moderately resistant or moderately susceptible, and are: Xindong 20, Shimai 19, Qingchun 5, Jining 13, Kaimai 21, and Keyi 26. These varieties can be used as breeding parents for genetic research or low-toxin wheat varieties based on their characteristics.

Claims

1. A method for evaluating the transport capacity of wheat ear tissue for DON toxin, characterized in that: The following steps are involved: 1) During the flowering stage of wheat ears, two florets of any spikelet from the 1st to the 6th spikelet from the top of the wheat ear were inoculated with spores of Fusarium graminearum; 2) According to the severity of the disease, the ears are divided into infected spikelets, transitional spikelets and non-infected spikelets. The infected spikelets are dry, no full seeds are formed, and the spike axis is browned. The seeds in the transitional spikelet are full, the husk is white, and the spike axis is not browned. The non-infected spikelet is no different from the normal spikelet, with full grains. 3) The toxin content of the spikelets in the infected area is named DON IS The toxin content in the spikelets of the non-infected area is named DON US , the calculation formula for toxin transport capacity is established as: 4) When DTC≦0.2, the wheat is considered to have a low DON transport capacity; when DTC≧0.7, the wheat is considered to have a high DON transport capacity.

2. The method for evaluating the transport capacity of wheat ear tissue for DON toxin according to claim 1, characterized in that: Step 1) The concentration of the spore solution of Fusarium graminearum is 1×10 5 / mL, and each spikelet was inoculated with 10μL.

3. The method for evaluating the transport capacity of wheat ear tissue for DON toxin according to claim 1, characterized in that: The method for detecting the toxin content in the spikelets in the infected area in step 3) comprises: taking spike tissues of multiple independent wheat ears in the infected area, mixing them as a sample, grinding them into powder, and then detecting the toxin by liquid chromatography-mass spectrometry to obtain DON IS .

4. The method for evaluating the transport capacity of wheat ear tissue for DON toxin according to claim 1, characterized in that: Step 3) Collect ear tissues from multiple independent ears of wheat from non-infected areas and mix them as a sample, grind them into powder, and then detect the toxin by liquid chromatography-mass spectrometry to obtain DON. US .

5. Use of the method according to any one of claims 1 to 4 in gene localization or in screening wheat varieties with low toxin or low transport capacity.

6. The use according to claim 5, characterized in that: The low-toxin wheat variety is a wheat variety with DTC≦0.

2.

7. The use according to claim 6, characterized in that: The wheat varieties with low toxin or low transport capacity include Xindong 20, Shimai 19, Qingchun 5, Jining 13, Kaimai 21, and Keyi 26.

8. A method for screening low-toxin wheat varieties or low-transport capacity wheat varieties, characterized in that: The following steps are involved: The method according to claim 1 is used to screen wheat varieties with DTC ≤ 0.

2.

9. The method for screening low-toxin wheat varieties or low-transport capacity wheat varieties according to claim 8, characterized in that: The wheat varieties with low toxin or low transport capacity include Xindong 20, Shimai 19, Qingchun 5, Jining 13, Kaimai 21, and Keyi 26.

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