Molecular marker for resistance of panonychus citri to ethyl azamite, its application and detection method

Abstract

The application discloses a molecular marker of citrus full mite resistance to ethyl acetazolamide and application thereof, and the molecular marker is SNP mutation A269G and / or T270A of succinate dehydrogenase SDHD subunit of the citrus full mite. A detection method of the molecular marker of the citrus full mite resistance to ethyl acetazolamide is also disclosed, total DNA of the citrus full mite to be detected is extracted, PCR amplification is carried out with the total DNA as a template, a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region is obtained, and the obtained gene fragment is subjected to sequence comparison with the molecular marker, so that the resistance of the citrus full mite to be detected to ethyl acetazolamide can be judged. The molecular marker and the detection method can simultaneously detect a large number of samples, can quickly and effectively detect whether the resistance SNP molecular marker and the mutation proportion exist in a field population of the citrus full mite, and can evaluate the mutation frequency of the corresponding site in a certain geographical population.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and relates to a molecular marker, in particular to a molecular marker for resistance of Panonychus citri to cyenpyrafen and an application and detection method thereof. BACKGROUND

[0002] Panonychus citri, also known as citrus red mite, belongs to Arachnida, Trombidiformes, Tetranychidae and Panonychus, and is a worldwide citrus mite, which is distributed in China, Japan, the United States, Europe and other countries (Barbar, 2014; Gotoh et al., 2003; Mcgegor, 1948). Panonychus citri damages citrus trees by feeding on the tender shoots, leaves and fruit skin with its mouthparts, causing the damaged parts to appear gray spots or even lose all green, affecting the photosynthesis of the fruit trees and limiting the vegetative growth of the fruit trees. In severe cases, it can cause defoliation and fruit drop, affecting the yield and quality of citrus. It is a key mite that needs to be monitored and controlled in citrus production areas in China every year (Xia et al., 2014).

[0003] Cyenpyrafen (Cyetpyrafen, SYP-9625), molecular formula C 24 H 31 N3O2, chemical name (Z)-2-(4-tert-butylphenyl)-2-cyano-1-(1-ethyl-3-methylpyrazol-5-yl) vinyl-2,2-dimethylpropanoate, CAS registration number: 1253429-01-4, belongs to mitochondrial complex II inhibitors, and the target is mitochondrial respiratory chain complex II, also known as succinate dehydrogenase (EC 1.3.5.1) or succinate-ubiquinone oxidoreductase (SQR). Cyenpyrafen disrupts the function of mitochondrial protein complex II, hinders electron transfer, destroys the oxidative phosphorylation process, and prevents the energy utilization of mites, thereby paralyzing and killing the mites (De Rouco et al., 2023). It shows strong acaricidal activity to various age stages of Panonychus citri, Tetranychus cinnabarinus, Tetranychus urticae and other plant mites through contact and stomach poisoning.

[0004] According to the current reports, the SDH mutations related to complex II inhibitor resistance are H258Y / L and I260T / V on the SDHB subunit, S56L (Q56L) on the SDHC subunit, and I263T on the SDHD subunit. et al., 2022; Njiru et al., 2022; Sugimoto et al., 2020). The co-occurrence of SDHB-I260V and SDHC-S56L mutations can confer a higher level of resistance to pyflubumide and kniprtript in the spider mites than single mutation (Maeoka and Osakabe, 2021). A recent report indicated that the resistance trait of T. urticae to etoxazole is incompletely dominant, and SDHB-I260V and SDHD-R119C mutations were found in the etoxazole-resistant strain of T. urticae, and it was confirmed by constructing near-isogenic lines that the presence of SDHB-I260V alone can lead to high-level resistance of T. urticae to etoxazole, in addition, the simultaneous mutation of SDHB-I260V and SDHD-R119C can contribute significantly to the production of extremely high-level resistance (Sun Jingyu, 2022; Li Chunji, 2023). In addition, there is no other related research report on etoxazole target resistance. SUMMARY

[0005] The purpose of the present application is to solve the above problems, and provide a molecular marker for etoxazole resistance of citrus red mite and its application and detection method.

[0006] In order to achieve the purpose of the present application, the technical scheme adopted by the present application is:

[0007] The first aspect of the present application provides a molecular marker for etoxazole resistance of citrus red mite, the molecular marker is the sequence after SNP mutation of succinate dehydrogenase SDHD subunit of citrus red mite, the CDS sequence of succinate dehydrogenase SDHD subunit gene of citrus red mite is shown as SEQ ID No: 30, and the SNP mutation is A269G and / or T270A mutation of the sequence shown as SEQ ID No: 30.

[0008] The SNP mutation A269G causes the 90th amino acid to be mutated from aspartic acid to glycine; and the SNP mutation T270A causes the 90th amino acid to be mutated from aspartic acid to glutamic acid.

[0009] The second aspect of the present application provides a primer pair for the above-mentioned molecular marker, the primer pair is SDHD-F / SDHD-R, and the primer sequence is:

[0010] SDHD-F: 5'-TCAAATCTCACCGAACTCAA-3';

[0011] SDHD-R: 5'-GGTTGATGTTTGTTCCGATT-3'.

[0012] The third aspect of the present application provides a kit of the molecular marker, comprising the primer pair.

[0013] Preferably, the kit further comprises DNA extraction reagents and PCR amplification reagents.

[0014] The fourth aspect of the present application provides the use of the molecular marker, the primer pair or the kit in detecting the resistance of Panonychus citri to ethyl azamidofen.

[0015] The fifth aspect of the present application provides a method for detecting the resistance of Panonychus citri to ethyl azamidofen, comprising the following steps: extracting total DNA from Panonychus citri to be tested, using the total DNA as a template, and performing PCR amplification with the primer pair to obtain a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region, and then performing sequence alignment between the obtained gene fragment and the molecular marker of Panonychus citri to ethyl azamidofen, so as to determine the resistance of Panonychus citri to ethyl azamidofen.

[0016] If the sequence of the gene fragment obtained by PCR amplification is the sequence of the molecular marker, it is determined that the Panonychus citri to be tested has resistance to ethyl azamidofen.

[0017] Preferably, the reaction system of the PCR amplification is as follows: 25 μL of 2×PhantaMax Master Mix, 2 μL of each of the forward and reverse primers with a concentration of 10 μM, 2 μL of the DNA template of the sample to be tested, and supplementing with ddH2O to 50 μL.

[0018] Preferably, the amplification procedure of the PCR is as follows: pre-denaturation at 95℃ for 3 min, then denaturation at 95℃ for 15 sec, annealing at 56℃ for 15 sec, extension at 72℃ for 30 sec, 40 cycles, and finally extension at 72℃ for 5 min.

[0019] The present application has the following beneficial effects:

[0020] 1) The present application discovers a molecular marker of the resistance of Panonychus citri to ethyl azamidofen, which is verified in field populations. The marker has high specificity and stability. The method of the present application can simultaneously detect a large number of samples, and can quickly and effectively detect whether the field population of Panonychus citri has the resistant SNP molecular marker and the mutation proportion, and can quickly and effectively evaluate the mutation frequency of the corresponding site in a certain geographical population. Through the analysis of the mutation frequency of the molecular marker of the sample to be tested, it can be further known whether the sample to be tested is a sensitive strain or a resistant strain of ethyl azamidofen, and the resistance level of Panonychus citri in the field can be monitored at any time, so that the management of pest resistance can be done well. The present application has important significance for reducing the use of chemical pesticides, environmental protection, and sustainable development of orchards.

[0021] 2) The present application provides specific forward and reverse primers of the molecular marker of the resistance of Panonychus citri to ethyl acetate, and also provides an optimized PCR system and amplification procedure, which can successfully and rapidly amplify a 941 bp DNA band from Panonychus citri, and the band is closely related to the resistance of Panonychus citri to ethyl acetate, and the method is simple and fast in operation, and has high detection specificity and sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the analysis of the conserved domains of the four subunits (PcSDHA, PcSDHB, PcSDHC and PcSDHD) of succinate dehydrogenase.

[0023] Figure 2 is the sequencing results of the SNP of the PcSDHD gene of Panonychus citri of the sensitive strain, the resistant strain and the field population in Yuxi, Yunnan.

[0024] Figure 3 is the nonsynonymous mutation of the PcSDHD gene of Panonychus citri of the resistant strain and the sensitive strain.

[0025] Figure 4 is the schematic diagram of the location of the SNP mutation site of the succinate dehydrogenase SDHD subunit of Panonychus citri in the genome. DETAILED DESCRIPTION

[0026] The present application will be further described below in conjunction with examples, but the present application is not limited by the examples.

[0027] In the following examples, the experimental methods are conventional methods unless otherwise specified.

[0028] Main experimental instruments: glass slide, scissors, dissection needle, sponge, culture dish, artist's pen, cotton thread, filter paper, body microscope, light incubator (SANYO), Burkard spray tower (Burkard Manufacturing, Co Ltd).

[0029] Main reagent sources:

[0030] Ethyl acetate suspension agent (30% of active ingredient content, Shenyang Kechuang Chemical Co., Ltd.),

[0031] 2x PhantaMax MasterMix (Nanjing Novozyme Biological Technology Co., Ltd., item number: P515),

[0032] DNA Micro Kit cell / tissue DNA extraction kit (Qiagen, Germany),

[0033] Tissue Ex-Amp PCR Kit (Applied Biological Materials Inc, Vancouver, Canada, Cat# G927).

[0034] Example 1

[0035] I. Experimental Methods

[0036] (1) Source of Test Mites

[0037] Citrus red mite susceptible strain (SS): The original population of citrus red mite in the laboratory was collected from the un-applied pesticide for many years on the fruit of Seville orange in the orchard of Citrus Research Institute (Chongqing, Beibei) in 2005. It was continuously subcultured in the laboratory without pesticide exposure and was fed with Seville orange until now. It was considered as a susceptible strain to acaricides.

[0038] Citrus red mite YF-resistant strain (CyetR-YF): It was collected from Yongfu region of Guangxi Zhuang Autonomous Region and was continuously sprayed with YF in the laboratory to maintain resistance.

[0039] (2) Toxicity test on female adults

[0040] The leaf disc method was used to conduct indoor bioassay on female adults of citrus red mite. Flat citrus leaves were collected and placed in a culture dish with the leaf surface facing down (supported by a water-containing sponge). The leaves were surrounded with fine cotton thread to prevent the mites from escaping, and the thread ends were covered with absorbent cotton to keep the thread moist. 30 healthy and active female adults were selected and placed on each leaf. Based on the preliminary test, YF suspensions were prepared at 5 concentrations for treatment. The feeding device was placed under the Potter spray tower, and 1 mL of the pesticide was sprayed. After being taken out and dried, it was placed in a constant temperature and light incubator with a temperature of 25±1℃, a relative humidity of 70%-80%, and a light cycle of 14L:10D. After 24 hours, the mortality of female adults in each group was observed and counted under a body microscope. When the mortality of female adults in the control group was <10%, it was considered as an effective experiment. The Polo plus 2.0 software was used to calculate the LC50, 95% confidence interval, and correlation coefficient. The control group was treated with water, and each concentration treatment was repeated 3 times.

[0041] (3) Searching for succinate dehydrogenase gene in the genome of citrus red mite

[0042] The succinate dehydrogenase gene (SDH) was searched in the internal genome annotation database of citrus red mite in the unit. The MEGA7.0 software was used for phylogenetic analysis and classification of the gene. The structure of the succinate dehydrogenase gene was analyzed by gene structure annotation.

[0043] (4) Screening of resistance vs. susceptible SNPs

[0044] SNPs (non-synonymous mutations) between Cy. citri ethaboxam-resistant strain (CyetR-YF) and susceptible strain (SS) were compared by Sanger sequencing.

[0045] Two strains were selected 200 mites each, and the total DNA of each strain was extracted by DNA Micro Kit according to the manufacturer's instructions. The four subunits (PcSDHA, PcSDHB, PcSDHC and PcSDHD) of the succinate dehydrogenase gene were amplified by PCR using high-fidelity enzyme 2x PhantaMax Master Mix and specific primers. Since the amplification bands of PcSDHA and PcSDHB were long, the experiment was carried out by segment cloning, and a certain length of repetitive sequence was set between each segment to facilitate the splicing of sequencing results. The PCR amplification primers are shown in Table 1. The amplification products were subjected to Sanger sequencing, the sequencing peak spectrum was analyzed by SnapGene software, and the base and amino acid sequences were aligned by BioXM2.7 to screen for double peaks, non-synonymous mutations, etc.

[0046] Table 1 PCR primers of Cy. citri succinate dehydrogenase gene

[0047]

[0048]

[0049] (5) Genotype identification of single mite in field population

[0050] The field population of Cy. citri was collected from Meishan, Sichuan (SC-MS) and Yuxi, Yunnan (YN-YX). The collected Cy. citri population was first subjected to indoor bioassay and the population resistance fold (LC 50 value compared with the susceptible strain) was calculated, and the specific method steps refer to the method of the previous step "(2) Toxicity test of female adult mites".

[0051] After PCR sequence alignment of citrus red mite ethaboxam-resistant and sensitive populations, the SNPs found were used as candidate sites, and field populations of citrus red mite were selected again to further sequence verification of candidate SNPs using single mite genomic DNA as a template. The population of each region was taken in an appropriate amount of citrus red mite, and the single mite genome was extracted using Tissue Ex-Amp PCR Kit initial sample one-step direct PCR kit. The method is as follows: about the right amount of citrus red mite was picked, each mite was placed in a 200 μL PCR tube, and the sample was ground with a sterile toothpick, then 20 μL of Tissue Ex-Amp PCR Kit lysis solution was added, and the PCR instrument was placed at 55°C for 10 min and 95°C for 5 min. After the sample was treated, 2 μL was taken as the DNA template. The PCR reaction system was: 2x Phanta Max Master Mix 25 μL, 10 μM concentration of forward and reverse primers 2 μL each, sample DNA template 2 μL, supplemented with ddH2O to 50 μL. The PCR amplification program was: 95°C pre-denaturation for 3 min, then 95°C denaturation for 15 sec, 56°C annealing for 15 sec, 72°C extension for 30 sec, 40 cycles; finally 72°C extension for 5 min. The PCR specific amplification primer was: EVM0001559 gene was amplified using SDHD-F / SDHD-R primer. The amplification product was subjected to Sanger sequencing to detect the genotype and sequencing peak of the candidate site.

[0052] (6) Mutation frequency analysis

[0053] According to the sequencing results, the genotype of the field population was analyzed, counted, and compared with the sensitive population and the resistant population, and the mutation frequency was calculated as follows:

[0054]

[0055] II. Results and analysis

[0056] (1) Toxicity test results

[0057] Table 2 Toxicity test of ethaboxam on sensitive strain (SS) and resistant strain (CyetR-YF) of citrus red mite

[0058]

[0059] The toxicity test results of ethaboxam on female adult mites of sensitive and resistant strains of citrus red mite showed that the LC 50 value of ethaboxam on the sensitive strain was 0.91 mg / L -1 , and the LC 50 value of ethaboxam on the resistant strain was 59448.85 mg / L -1The resistance fold of the resistant line reached 65328.40 times, reaching a high resistance level.

[0060] (2) Screening of citrus red mite succinate dehydrogenase genes

[0061] We screened the citrus red mite gene annotated genes, and found 4 subunit genes of succinate dehydrogenase, respectively EVM0002719 gene (PcSDHA, its sequence is shown as SEQ ID No: 27), EVM0002899 gene (PcSDHB, its sequence is shown as SEQ ID No: 28), EVM0009234 gene (PcSDHC, its sequence is shown as SEQ ID No: 29) and EVM0001559 gene (PcSDHD, its CDS sequence is shown as SEQ ID No: 30) (Table 3). Consistent with the reported number of mite succinate dehydrogenase genes. The amplification product fragment of the primer pair SDHD-F / SDHD-R of the PcSDHD gene is 941 bp, the sequence is shown as SEQ ID NO. 31, and the schematic diagram of the SNP mutation site in it is shown as Figure 4

[0062] Table 3 Four subunit genes of succinate dehydrogenase contained in the genome of citrus red mite

[0063]

[0064] Bioinformatics analysis was performed on the sequences of PcSDHA, PcSDHB, PcSDHC and PcSDHD genes, as shown in Table 3, the open reading frame lengths of the four subunits were 1968 bp, 915 bp, 528 bp and 423 bp, respectively, encoding 655, 304, 175 and 140 amino acids. The relative molecular weight was 72.64 kDa, 34.40 kDa, 18.77 kDa and 15.71 kDa, respectively, and the total relative molecular weight of mitochondrial complex II was 141.52 kDa. PcSDHA and PcSDHB were hydrophilic, and PcSDHC and PcSDHD were hydrophobic. The results of the analysis of the conserved domains of each subunit showed (such as Figure 1 ​), the 38th-655th amino acid fragment of PcSDHA is the typical conserved structure region of succinate dehydrogenase flavoprotein subunit "PTZ00139". The 58th-290th amino acid fragment of PcSDHB is the typical conserved structure region of succinate dehydrogenase iron-sulfur protein subunit "PLN00129". The 57th-173rd amino acid fragment of PcSDHC is the typical conserved structure region of succinate dehydrogenase C subunit "SQR_TypeC_SdhC", the 67th, 74th, 75th and 78th amino acids are proximal ubiquinone binding sites, and the 78th, 84th, 85th, 132nd and 133rd amino acids are proximal heme binding sites. The 43rd-136th amino acid fragment of PcSDHD is the typical conserved structure region of succinate dehydrogenase D subunit "SQR_TypeC_CybS", the 91st amino acid is a proximal ubiquinone binding site, and the 79th and 83rd amino acids are proximal heme binding sites.

[0065] (3) Screening of SNPs of four subunits (PcSDHA, PcSDHB, PcSDHC and PcSDHD) of succinate dehydrogenase

[0066] Sanger sequencing was used to compare the SNPs of the four subunits (PcSDHA, PcSDHB, PcSDHC and PcSDHD) of succinate dehydrogenase between the resistant and sensitive lines. By comparison, no non-synonymous mutation was found in the PcSDHA, PcSDHB and PcSDHC genes, and a non-synonymous mutation occurred at the 90th amino acid of the SDHD subunit, and there were two mutant types, one of which was a conversion of the 269th base in the open reading frame (SEQ ID NO: 30), with A base in the sensitive line and G base in the resistant line, i.e. A269G mutation Figure 2 ), resulting in a mutation of the 90th amino acid from aspartic acid to glycine, i.e. D90G Figure 3 ; and the other was a transversion of the 270th base in the open reading frame (SEQ ID NO: 30), with T base in the sensitive line and A base in the resistant line, i.e. T270A mutation Figure 2 ), resulting in a mutation of the 90th amino acid from aspartic acid to glutamic acid, i.e. D90E Figure 3 .

[0067] (4) Toxicity test of ethyl (5-bromo-2-oxo-3-oxazolidinyl) acetate on field population of Panonychus citri

[0068] Table 4 Toxicity test of ethyl (5-bromo-2-oxo-3-oxazolidinyl) acetate on field population of Panonychus citri

[0069]

[0070] The virulence of *Paecilomyces citrus* in field populations in Sichuan Asbestos (CS-SM) and Yunnan Yuxi (YN-YX) was determined. The results showed that the LC50 of the Yunnan Yuxi population (YN-YX) was significantly lower. 50 =1097.94 mg / L, resistance multiple of 1206.53, similar to etoxazole-resistant strains, indicating extremely high resistance levels. LC50 of the Sichuan Asbestos population (CS-SM) 50 =6.85mg / L, resistance multiple is 7.52, indicating a low-resistance population.

[0071] (5) PcSDHD mutation frequency analysis

[0072] Table 5. Statistics on the mutation frequency of the PcSDHD gene in citrus pseudocoma in different regions.

[0073]

[0074] *Note: n is the number of *Pseudorasbora citrinum* sequences obtained using Sanger sequencing.

[0075] Twenty to thirty citrus psyllids from different regions were selected for single-headed mite PCR amplification and sequencing, and the mutation frequency of the 90th amino acid was calculated. As shown in Table 5, the PcSDHD amino acid sequence in the Sichuan Asbestos population (CS-SM) was consistent with the indoor sensitive strain (SS), and no sense mutations were found. All were GAT / GAT homozygous.

[0076] In the Yunnan Yuxi population (YN-YX), a mutation was found in the CDS sequence of PcSDHD, where the A at position 269 was changed to G, i.e., A269G. This caused a mutation at position 90 of PcSDHD, where the amino acid was changed from aspartic acid (D, Asp, codon GAT) to glycine (G, Gly, codon GGT). The mutation frequency of D90G was 54.35%. Three genotypes were found in the population: GAT / GAT, GGT / GGT, and GGT / GAT. Figure 2 ).

[0077] In the etoxazole-resistant strain (CyetR-YF), the mutation rate of amino acid position 90 in PcSDHD reached 100%, with two mutation types present. Specifically, the T at position 270 of the PcSDHD CDS sequence was replaced with an A (T270A), resulting in a mutation of aspartic acid (D, Asp) to glutamic acid (E, Glu, codon GAA); the A at position 269 of the PcSDHD CDS sequence was replaced with a G (A269G), resulting in a mutation of aspartic acid (D, Asp) to glycine (G, Gly). The mutation frequency of D90E was 97.92%, and that of D90G was 2.08%. Two genotypes, GAA / GAA and GAA / GGT, were present in the population. Figure 2). It showed that the mutation of the 90th amino acid had obvious correlation with resistance.

[0078] III. Analysis

[0079] On the basis of obtaining the citrus full claw mite ethylazocyclonol resistant strain, the SNP screening of the succinate dehydrogenase gene of the citrus full claw mite sensitive strain was carried out, and finally two non-synonymous mutations A269G (D90G) and T270A (D90E) were found in the PcSDHD gene of the resistant citrus full claw mite. Subsequently, field populations from two regions were collected, and single mite PCR amplification and sequencing were carried out to verify the correlation between the mutation sites and ethylazocyclonol resistance. The results showed that the two mutant types of PcSDHD gene A269G (D90G) and T270A (D90E) showed high correlation with drug resistance.

[0080] Succinate dehydrogenase, also known as mitochondrial complex II or succinate-ubiquinone oxidoreductase, is a transmembrane protein complex composed of four subunits. SDHA (flavoprotein, FP) and SDHB (iron-sulfur protein, IP) form a water-soluble heterodimer with succinate dehydrogenase catalytic activity; SDHC and SDHD are anchored on the inner membrane of mitochondria to form a transmembrane part with ubiquinone reductase catalytic activity (Ackrell, 2000; Szeto et al., 2007; Yankovskaya et al., 2003). It is a key enzyme involved in intracellular oxidative phosphorylation electron transport chain and tricarboxylic acid cycle, and is also the action target of commonly used fungicides, new pyridine acrylonitrile acaricides (IRAC: 25A: cyenopyrafen, cyflumetofen and ethylazocyclonol) and formanilide acaricides Pyfubumide (IRAC: 25B) (Fotoukkiaii et al., 2020; Umetsu and Shirai, 2020). Research reports indicate that mutations in the SDHB subunit and the SDHC subunit are associated with resistance to such acaricides, among which the SDHB subunit I260T / V is associated with cyflumetofen resistance, and the SDHB subunit I260V and the SDHC subunit S56L mutation linkage appear to be associated with cyenopyrafen and pyflubumide resistance (Maeoka and Osakabe, 2021; Sugimoto et al., 2020), and H258Y / L mutations can cause the production of cyenopyrafen and pyflubumide cross-resistance et al., 2022; Njiru et al., 2022). Sun Jingyu and Li Chunji found SDHB-I260V and SDHD-R119C mutations in the T. urticae ethaboxam-resistant strain, and confirmed that SDHB-I260V alone can cause high-level resistance of T. urticae to ethaboxam, and the coexistence of SDHB-I260V and SDHD-R119C mutations can contribute to the development of very high-level resistance (Sun Jingyu, 2022; Li Chunji, 2023). We analyzed the sequences of each subunit of mitochondrial complex II in the laboratory susceptible strain (SS) and ethaboxam-resistant strain (CyetR-YF) of C. citricola, and found that the corresponding mutation types in SDHB and SDHC subunits in T. urticae did not occur, and the D90E mutation in the SDHD subunit was not a homologous mutation of SDHD-R119C in T. urticae.

[0081] Ethaboxam, as a mitochondrial complex II inhibitor acaricide, can act on each mite stage, disrupt the function of mitochondrial protein complex II, hinder electron transfer, destroy the oxidative phosphorylation process, and prevent the energy utilization of pest mites, thereby paralyzing and killing pest mites (De Rouck et al., 2023). However, the specific mode of action of the target succinate dehydrogenase mutation that reduces the sensitivity of C. citricola to ethaboxam has not been reported.

[0082] The present application found that the mutation of the 90th amino acid of the succinate dehydrogenase SDHD subunit of C. citricola is highly correlated with the resistance trait in the field, but the specific mode of action of the target succinate dehydrogenase mutation that reduces the sensitivity of C. citricola to ethaboxam has not yet been determined. Researchers can further explore the influence of the mutation of the 90th amino acid of the succinate dehydrogenase SDHD subunit on the binding of SDH and ethaboxam, and the role of PcSDHD gene mutation in resistance through reverse genetics methods based on the research results of the present application.

Claims

1. A molecular marker for resistance of Panonychus citri to clofentezine, characterized in that: The molecular marker is the sequence of the SNP mutation of the CDS of the succinate dehydrogenase SDHD subunit gene of the citrus full mite, wherein the CDS sequence is shown as SEQ ID No: 30, and the SNP mutation is the mutation of A269G or T270A in the sequence shown as SEQ ID No:

30.

2. The molecular marker of claim 1, wherein: The SNP mutation A269G causes the 90th amino acid to be mutated from aspartic acid to glycine; and the SNP mutation T270A causes the 90th amino acid to be mutated from aspartic acid to glutamic acid.

3. The use of the molecular marker in claim 1 in detecting the resistance of the citrus full mite to ethaziocyclon.

4. A method for detecting a molecular marker of resistance to ethyl (Z)-3-ethoxy-2- methylpropenyl N- cyanothioacetimidate in Panonychus citri, characterized by: First, total DNA of the citrus full mite to be tested is extracted, and then PCR amplification is performed on the total DNA as a template and with a primer pair to obtain a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region, and the obtained gene fragment is subjected to sequence alignment with the molecular marker of the citrus full mite to ethaziocyclon in claim 1, so as to determine the resistance of the citrus full mite to be tested to ethaziocyclon; the primer pair is SDHD-F / SDHD-R, and the primer sequences are as follows: SDHD-F: 5'-TCAAATCTCACCGAACTCAA-3'; SDHD-R: 5'-GGTTGATGTTTGTTCCGATT-3'.

5. The method of claim 4, wherein: If the sequence of the gene fragment obtained by PCR amplification is the sequence of the molecular marker in claim 1, it is determined that the citrus full mite to be tested has resistance to ethaziocyclon.

6. The method of claim 4, wherein: The reaction system of the PCR amplification is as follows: 2 × Phanta Max Master Mix 25 μL, 2 μL of each of forward and reverse primers with a concentration of 10 μM, 2 μL of a DNA template of a sample to be tested, and supplemented with ddH2O to 50 μL.

7. The method of claim 6, wherein: The amplification program of the PCR is as follows: pre-denaturation at 95℃ for 3 min, then denaturation at 95℃ for 15 sec, annealing at 56℃ for 15 sec, extension at 72℃ for 30 sec, 40 cycles, and finally extension at 72℃ for 5 min.

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