Molecular marker for resistance of panonychus citri to etoxazole nitrile as well as application and detection method of molecular marker

Abstract

The invention discloses a molecular marker of resistance of panonychus citri to etoxazole nitrile and application of the molecular marker. The molecular marker is obtained by SNP (Single Nucleotide Polymorphism) mutation A269G and / or T270A of a panonychus citri succinate dehydrogenase SDHD subunit. The invention also discloses a detection method of the molecular marker for the resistance of the panonychus citri to the etoxazole nitrile, which comprises the following steps: firstly, extracting the total DNA of the panonychus citri to be detected, carrying out PCR amplification by taking the total DNA as a template to obtain a succinate dehydrogenase SDHD subunit gene segment containing a mutation region, and carrying out sequence comparison on the obtained gene segment and the molecular marker to obtain the resistance of the panonychus citri to the etoxazole nitrile. The resistance of the panonychus citri to be detected to the SYP-9625 can be judged. According to the molecular marker and the detection method, a large number of samples can be detected at the same time, whether the resistance SNP molecular marker exists in a field population of the panonychus citri or not and the mutation proportion can be rapidly and effectively detected, and the mutation frequency of a corresponding site in a certain geographical population is evaluated.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a molecular marker, in particular to a molecular marker of citrus mites' resistance to ethidium bromide and an application and detection method thereof. Background Art

[0002] Panonychus citri, also known as citrus red spider, belongs to the Arachnida, Trombidiformes, Tetranychidae, and the genus Panonychus. It is a worldwide citrus pest mite, distributed in China, Japan, the United States, Europe and other countries (Barbar, 2014; Gotohe et al., 2003; McGegor, 1948). Panonychus citri harms citrus trees with juvenile mites, nymphs and adults. It mainly sucks the juice of tender shoots, leaves and fruit skin through its mouthparts, causing gray-white spots or even complete loss of green on the affected parts, affecting the photosynthesis of fruit trees and limiting the nutritional growth of fruit trees. In severe cases, it can cause leaf and fruit fall, affecting the yield and quality of citrus. It is a pest mite that is monitored and controlled every year in my country's citrus producing areas (Xia et al., 2014).

[0003] Cyetpyrafen (SYP-9625), molecular formula C 24 H 31 N 3 O 2 , chemical name (Z)-2-(4-tert-butylphenyl)-2-cyano-1-(1-ethyl-3-methylpyrazol-5-yl) vinyl-2,2-dimethylpropionate, CAS registration number: 1253429-01-4, belongs to mitochondrial complex II inhibitors, and its target is mitochondrial respiratory chain complex II, also known as succinate dehydrogenase (EC 1.3.5.1) or succinate-ubiquinone oxidoreductase (SQR). Ethiofenamide disrupts the function of mitochondrial protein complex II, hinders electron transfer, destroys the oxidative phosphorylation process, and prevents the energy utilization of mite cells, thereby paralyzing and killing the mite (DeRocketal., 2023). It mainly shows strong acaricidal activity against various plant pests such as citrus mites, cinnabarinus mites, two-spotted mites and apple mites of all ages through contact and stomach poisoning.

[0004] According to current reports, SDH mutations associated with resistance to complex II inhibitors include H258Y / L and I260T / V on the SDHB subunit, and S56L ( et al., 2022; Njiru et al., 2022; Sugimoto et al., 2020). The co-occurrence of SDHB-I260V and SDHC-S56L mutations can confer higher resistance levels to pyflubumide and cypermethrin than single mutations (Maeoka and Osakabe, 2021). Recent reports indicate that the resistance of two-spotted spider mites to ethidium bromide is an incomplete dominant inheritance. SDHB-I260V and SDHD-R119C mutations were found in two-spotted spider mites ethidium bromide-resistant strains, and the construction of near-isogenic lines confirmed that the presence of SDHB-I260V alone can lead to high-level resistance of two-spotted spider mites to ethidium bromide. In addition, the simultaneous mutation of SDHB-I260V and SDHD-R119C can make an important contribution to the generation of its extremely high level of resistance (Sun Jingyu, 2022; Li Chunji, 2023). Apart from this, there are no other reports on research on resistance to ethidium bromide. Summary of the invention

[0005] The object of the present invention is to provide a molecular marker for the resistance of Panonychus citri to ethidium bromide and its application and detection method in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a molecular marker for the resistance of Panonychus citri to ethidium bromide, wherein the molecular marker is a sequence after a SNP mutation occurs in the succinate dehydrogenase SDHD subunit of Panonychus citri, the CDS sequence of the succinate dehydrogenase SDHD subunit gene of Panonychus citri is shown in SEQ ID No: 30, and the SNP mutation is an A269G and / or T270A mutation in the sequence shown in SEQ ID No: 30.

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

[0009] The second aspect of the present invention provides a primer pair for the above-mentioned molecular marker, wherein 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 invention provides a molecular labeling kit as described above, comprising the primer pair as described above.

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

[0014] The fourth aspect of the present invention provides the use of the above-mentioned molecular marker or the above-mentioned primer pair or the above-mentioned kit in detecting the resistance of Panonychus citri to ethidium bromide.

[0015] The fifth aspect of the present invention provides a method for detecting the molecular marker of the resistance of citrus panonychus mites to ethtozoxanil: first extract the total DNA of the citrus panonychus mites to be tested, use the total DNA as a template, and perform PCR amplification with the above-mentioned primer pair to obtain a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region, and perform sequence comparison between the obtained gene fragment and the above-mentioned molecular marker of citrus panonychus mites to ethtozoxanil to determine the resistance of the citrus panonychus mites to ethtozoxanil.

[0016] If the gene fragment sequence obtained by PCR amplification is the sequence of the above-mentioned molecular marker, it is determined that the citrus mites to be tested are resistant to ethidium bromide.

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

[0018] Preferably, the PCR amplification program is: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, extension at 72°C for 30 sec, 40 cycles; and finally extension at 72°C for 5 min.

[0019] The beneficial effects of the present invention are:

[0020] 1) The present invention has discovered a molecular marker for the resistance of Panonychus citri to ethidium cypermethrin and has been verified in field populations. The marker has strong specificity and high stability. The method of the present invention can detect a large number of samples at the same time, can quickly and effectively detect whether the field population of Panonychus citri has the resistance SNP molecular marker and mutation ratio, can quickly and effectively evaluate the mutation frequency of the corresponding site in a certain geographical population, and through the mutation frequency analysis of the molecular marker of the sample to be tested of Panonychus citri, it can be further known whether the sample to be tested is a sensitive strain or a resistant strain to ethidium cypermethrin, and the resistance level of Panonychus citri in the field can be monitored at any time, and the work of controlling the resistance of harmful mites can be done well, which is of great significance for reducing the use of chemical pesticides, environmental protection, and sustainable development of orchards.

[0021] 2) The present invention provides specific forward and reverse primers for molecular markers of citrus mites' resistance to ethazilazopyr, and also provides an optimized PCR system and amplification program, which can successfully and quickly amplify a 941bp DNA band from citrus mites, and the band is a sequence closely related to citrus mites' resistance to ethazilazopyr. The method is simple and quick to operate, and has strong detection specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] Figure 2 These are the SNP sequencing results of the PcSDHD gene of the citrus mites in sensitive strains, resistant strains and field populations in Yuxi, Yunnan.

[0024] Figure 3 It is a non-synonymous mutation of the PcSDHD gene in the resistant and sensitive strains of citrus mites.

[0025] Figure 4 This is a 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 invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0028] Main experimental instruments: glass slides, scissors, dissecting needles, sponges, culture dishes, art pens, cotton thread, filter paper, stereo microscope, light incubator (SANYO), and Porter spray tower (Burkard Manufacturing, Co Ltd).

[0029] Main reagent sources:

[0030] Ethiophanate-methyl suspension (active ingredient content 30%, Shenyang Kechuang Chemicals Co., Ltd.),

[0031] 2×PhantaMax MasterMix (Nanjing Novozyme Biotechnology Co., Ltd., catalog number: P515),

[0032] DNA Micro Kit Cell / Tissue DNA Extraction Kit (Qiagen, Germany),

[0033] Tissue Ex-Amp PCR Kit: One-step direct PCR kit for primary samples (Applied Biological Materials Inc, Vancouver, Canada, Cat. No. G927).

[0034] Example 1

[0035] 1. Experimental Methods

[0036] (1) Test mite source

[0037] The citrus Panonychus citrus mite sensitive strain (SS): The original population of citrus Panonychus citrus mite in the laboratory was collected in 2005 from the orange peel that had not been sprayed with pesticides for many years beside the orchard of the Citrus Research Institute (Beibei, Chongqing). It was not exposed to pesticides in the laboratory and was continuously subcultured with orange peel to date. It is considered a mite-sensitive strain.

[0038] The citrus mites ethzofen-resistant strain (CyetR-YF) was collected from Yongfu, Guangxi Zhuang Autonomous Region, and ethzofen-resistant was continuously sprayed indoors to maintain resistance.

[0039] (2) Toxicity determination of female adult mites

[0040] The leaf disc method was used to carry out indoor bioassay of female adult mites of Panonychus citri. Flat citrus leaves were collected and placed face down in a petri dish (with a sponge containing water as a support). The leaves were wrapped with fine cotton thread to prevent adult mites from escaping. The ends of the thread were covered with absorbent cotton to keep the thread moist. Thirty healthy and vigorous female adult mites were grafted on each leaf. On the basis of the preliminary experiment, 5 concentrations of ethazofen suspension were prepared. The breeding device was placed under a Porter spray tower and sprayed with 1 mL of the agent. After being taken out and dried in the shade, it was placed in a constant temperature and light incubator with a temperature of 25±1℃, a relative humidity of 70% to 80%, and a photoperiod of 14L:10D. After 24 hours, the death of female adult mites in each group was observed and counted under a stereo microscope. When the death rate of female adult mites in the control group was <10%, it was considered a valid experiment. LC50, 95% confidence interval and correlation coefficient were calculated using Polo plus 2.0 software. Clear water treatment was used as control, and each concentration treatment was repeated 3 times.

[0041] (3) Searching for the succinate dehydrogenase gene of Panonychus citri in the genome

[0042] The succinate dehydrogenase gene (SDH) was retrieved from the internal Panonychus citri genome annotation database of our unit, and the phylogenetic analysis and classification of the gene were performed using MEGA7.0 software. The structure of the succinate dehydrogenase gene was analyzed through gene structure annotation.

[0043] (4) Resistance vs. sensitive SNPs screening

[0044] Sanger sequencing was used to compare the SNPs (non-synonymous mutations) in the succinate dehydrogenase gene between the ethidium bromide-resistant strain (CyetR-YF) and the susceptible strain (SS) of Panonychus citri.

[0045] 200 mites were selected from each of the two strains and The total DNA of both strains was extracted using a DNA Micro Kit, and the operation steps were carried out according to the instructions of the reagent. Using DNA as a template, the four subunits (PcSDHA, PcSDHB, PcSDHC, and PcSDHD) were PCR amplified using the high-fidelity enzyme 2×PhantaMax Master Mix and succinate dehydrogenase gene-specific amplification primers. Since the amplified bands of PcSDHA and PcSDHB were long, the experiment was carried out in a segmented cloning manner, and a certain length of repeat sequences was set between each segment to facilitate the splicing of sequencing results. The PCR amplification primers are shown in Table 1. The amplified products were sequenced by Sanger sequencing, and the sequencing peak spectrum was analyzed using SnapGene software. The base and amino acid sequences were aligned using BioXM2.7 to screen for double peaks in the bases and non-synonymous mutations.

[0046] Table 1 PCR primers for succinate dehydrogenase gene of Panonychus citri

[0047]

[0048]

[0049] (5) Identification of single-headed mite genotypes in field populations

[0050] Field populations of Panonychus citri were collected from Meishan, Sichuan (SC-MS) and Yuxi, Yunnan (YN-YX). The collected Panonychus citri populations were first subjected to indoor bioassays and the population resistance multiples were calculated (compared to the LC of the sensitive strain). 50 For specific methods and steps, refer to the method of the previous step "(2) Determination of toxicity of female adult mites".

[0051] After the PCR sequence comparison of the citrus panonychus mites resistant to ethidium cypermethrin and susceptible populations, the SNPs found were used as candidate sites, and then the field population of citrus panonychus mites was selected, and the genomic DNA of the single mite was used as a template to further sequence and verify the candidate SNPs. An appropriate number of citrus panonychus mites were taken from the population in each region, and the genome of the single mite was extracted using the Tissue Ex-Amp PCR Kit primary sample one-step direct PCR kit. The method is as follows: pick an appropriate amount of citrus panonychus mites, place each mite in a 200μL PCR tube, grind the sample with a sterile toothpick, then add 20μL Tissue Ex-Amp PCRKit lysis buffer, place it in a PCR instrument, heat at 55℃ for 10min, and heat at 95℃ for 5min. After the sample is processed, take 2μL as a DNA template. The PCR reaction system is: 2×Phanta Max Master Mix 25μL, 2μL of forward and reverse primers at a concentration of 10μM each, 2μL of the DNA template of the sample to be tested, and supplement with ddH 2 O 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; and finally 72°C extension for 5 min. The PCR specific amplification primers were: EVM0001559 gene was amplified using SDHD-F / SDHD-R primers. The amplified 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 genotypes of the field population were analyzed and counted, and compared with the sensitive and resistant populations. The mutation frequency was calculated as follows:

[0054]

[0055] 2. Results and Analysis

[0056] (1) Toxicity test results

[0057] Table 2 Toxicity test of ethamiclostrobin to sensitive strain (SS) and resistant strain (CyetR-YF) of Panonychus citri

[0058]

[0059] The results of the toxicity test of ethidium bromide on female adult mites of sensitive and resistant strains of Panonychus citri showed that the LC 50 The value is 0.91 mg L -1 , LC of resistant strains 50 The value is 59448.85 mg L -1The resistance multiple of the resistant strain reached 65328.40 times, reaching a high resistance level.

[0060] (2) Screening of succinate dehydrogenase gene of Panonychus citri

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

[0062] Table 3 The four subunit genes of succinate dehydrogenase contained in the genome of Panonychus citri

[0063]

[0064] Bioinformatics analysis of the gene sequences of PcSDHA, PcSDHB, PcSDHC and PcSDHD showed that the lengths of the open reading frames of the four subunits were 1968 bp, 915 bp, 528 bp and 423 bp, respectively, encoding 655, 304, 175 and 140 amino acids, respectively. The relative molecular weights were 72.64 kDa, 34.40 kDa, 18.77 kDa and 15.71 kDa, respectively, and the total relative molecular mass of mitochondrial complex II was 141.52 kDa. PcSDHA and PcSDHB are hydrophilic, while PcSDHC and PcSDHD are hydrophobic. The results of the conservative domain analysis of each subunit showed (e.g. Figure 1), the amino acid fragment of PcSDHA from 38 to 655 is a typical conserved structural region "PTZ00139" of the flavoprotein subunit of succinate dehydrogenase. The amino acid fragment of PcSDHB from 58 to 290 is a typical conserved structural region "PLN00129" of the iron-sulfur protein subunit of succinate dehydrogenase. The amino acid fragment of PcSDHC from 57 to 173 is a typical conserved structural region "SQR_TypeC_SdhC" of the C subunit of succinate dehydrogenase, the amino acids at positions 67, 74, 75, and 78 are proximal ubiquinone binding sites, and the amino acids at positions 78, 84, 85, 132, and 133 are proximal heme binding sites. The amino acid fragment of PcSDHD from 43 to 136 is a typical conserved structural region "SQR_TypeC_CybS" of the D subunit of succinate dehydrogenase, the amino acid at position 91 is a proximal ubiquinone binding site, and the amino acids at positions 79 and 83 are proximal heme binding sites.

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

[0066] Sanger sequencing was used to compare the SNPs in the genes of the four subunits of succinate dehydrogenase (PcSDHA, PcSDHB, PcSDHC, and PcSDHD) between the resistant strains and the sensitive strains. By comparison, it was found that there were no non-synonymous mutations in the PcSDHA, PcSDHB, and PcSDHC genes, and a non-synonymous mutation occurred at the 90th amino acid on 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 the sensitive strain being the A base and the resistant strain being the G base, namely the A269G mutation ( Figure 2 ), resulting in the mutation of the 90th amino acid from aspartic acid to glycine, namely D90G ( Figure 3 ); the other is a transversion at base 270 in the open reading frame (SEQ ID NO: 30), with the sensitive strain having a T base and the resistant strain having an A base, i.e., a T270A mutation ( Figure 2 ), resulting in the mutation of the 90th amino acid from aspartic acid to glutamic acid, namely D90E ( Figure 3 ).

[0067] (4) Toxicity test of ethamiclostrobin against field populations of Panonychus citri

[0068] Table 4 Toxicity test of ethamifenibacill to field population of Panonychus citri

[0069]

[0070] The toxicity test of field populations of Panonychus citri from Sichuan Shimian (CS-SM) and Yunnan Yuxi (YN-YX) showed that the LC 50 =1097.94 mg / L, the resistance multiple is 1206.53, which is the same as the ethazole-resistant strain, and is extremely high in resistance level. 50 =6.85mg / L, the resistance multiple is 7.52, which is a low-resistant population.

[0071] (5) PcSDHD mutation frequency analysis

[0072] Table 5 Statistics of mutation frequency of PcSDHD gene in citrus mite in different regions

[0073]

[0074] *Note: n is the number of Panonychus citri subjected to Sanger sequencing.

[0075] 20 to 30 Panonychus citri from different regions were selected for single-head mite PCR amplification and sequencing, and the mutation frequency of amino acid position 90 was calculated. As shown in Table 5, the amino acid sequence of PcSDHD in Sichuan asbestos population (CS-SM) was consistent with that of indoor sensitive strain (SS), no sense mutation was found, and all were GAT / GAT homozygous.

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

[0077] The mutation rate of amino acid at position 90 of PcSDHD in the ethidium bromide-resistant strain (CyetR-YF) reached 100%, and there were two types of mutations. The T at position 270 of the CDS sequence of PcSDHD was converted to A (i.e., T270A), resulting in the mutation of aspartic acid (D, Asp) to glutamic acid (E, Glu, codon is GAA); the A at position 269 of the CDS sequence of PcSDHD was converted to G (i.e., A269G), resulting in the mutation of aspartic acid (D, Asp) to glycine (G, Gly). The mutation frequency of D90E was 97.92%, and the mutation frequency of D90G was 2.08%. There were two genotypes in the population, GAA / GAA and GAA / GGT ( Figure 2). This indicates that the mutation of amino acid 90 is significantly correlated with resistance.

[0078] Analysis

[0079] Based on the obtained citrus panonychus resistant strains of ethidium cypermethrin, the present application conducted SNP screening on the succinate dehydrogenase gene of the citrus panonychus resistant strains, and finally found two non-synonymous mutations A269G (D90G) and T270A (D90E) in the PcSDHD gene in the resistant citrus panonychus mites. Subsequently, field populations from the two regions were collected, and single-headed mite PCR amplification and sequencing were performed to verify the connection between the mutation site and ethidium cypermethrin resistance. The results showed that the two mutant types of the PcSDHD gene A269G (D90G) and T270A (D90E) were highly correlated 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 the 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 the intracellular oxidative phosphorylation electron transport chain and tricarboxylic acid cycle, and is also the target of commonly used fungicides, new acrylonitrile acaricides (IRAC: 25A: cypermethrin, diflubenzuron and ethoxyfenoxam) and carboxanilide acaricide pyfubumide (IRAC: 25B) (Fotoukkiaii et al., 2020; Umetsu and Shirai, 2020). Studies have reported that mutations on the SDHB and SDHC subunits are associated with resistance to this type of acaricide, among which SDHB subunit I260T / V is associated with resistance to cyfluthrin, while the linked occurrence of SDHB subunit I260V and SDHC subunit S56L mutations is associated with resistance to cyfluthrin and pyflubumide (Maeoka and Osakabe, 2021; Sugimoto et al., 2020). The H258Y / L mutation can lead to the production of cross-resistance to cyfluthrin and pyflubumide ( et al., 2022; Njiru et al., 2022). Sun Jingyu and Li Chunji found SDHB-I260V and SDHD-R119C mutations in the two-spotted spider mite ethidium resistant strain, and confirmed by constructing near-isogenic lines that SDHB-I260V alone can lead to high-level resistance of two-spotted spider mites to ethidium sulfaquinoxaline, and the co-existence of SDHB-I260V and SDHD-R119C mutations can make an important contribution to the generation of extremely high levels of resistance (Sun Jingyu, 2022; Li Chunji, 2023). By comparing the sequences of each subunit of mitochondrial complex II in the indoor sensitive strain of citrus mites (SS) and the ethidium sulfaquinoxaline resistant strain (CyetR-YF), we did not find the corresponding mutation types in the two-spotted spider mite in the SDHB and SDHC subunits, and the D90E mutation on the SDHD subunit is not the homologous mutation of the two-spotted spider mite SDHD-R119C.

[0081] As a miticide of the mitochondrial complex II inhibitor type, ethazofop-butyl can act on all mite states, disrupting the function of mitochondrial protein complex II, hindering electron transfer, destroying the oxidative phosphorylation process, and preventing the energy utilization of mite cells, thereby paralyzing and killing the mite (De Roucket et al., 2023). However, there has been no relevant report on the specific mode of action of the target succinate dehydrogenase mutation that reduces the sensitivity of citrus mites to ethazofop-butyl.

[0082] The present invention found that the 90th amino acid mutation of the succinate dehydrogenase SDHD subunit of Panonychus citri was highly correlated with the resistance traits in the field, but the specific mode of action of the target mutation to reduce the sensitivity of Panonychus citri to ethtozofen has not yet been determined. Based on the research results of the present invention, researchers can further explore the effect of the 90th amino acid mutation of the succinate dehydrogenase SDHD subunit of the target on the binding ability of SDH and ethtozofen, and explore the role of PcSDHD gene mutation in resistance through reverse genetics methods.

Claims

1. A molecular marker for the resistance of Panonychus citri to ethidium bromide, characterized in that: The molecular marker is a sequence after a SNP mutation occurs in the succinate dehydrogenase SDHD subunit of Panonychus citri, the CDS sequence of the succinate dehydrogenase SDHD subunit gene of Panonychus citri is shown in SEQ ID No: 30, and the SNP mutation is an A269G and / or T270A mutation in the sequence shown in SEQ ID No:

30.

2. The molecular marker according to claim 1, characterized in that: The SNP mutation A269G causes the 90th amino acid to mutate from aspartic acid to glycine; the SNP mutation T270A causes the 90th amino acid to mutate from aspartic acid to glutamic acid.

3. A primer pair for detecting the molecular marker according to claim 1, characterized in that: The primer pair is SDHD-F / SDHD-R, and the primer sequence is: SDHD-F:5'-TCAAATCTCACCGAACTCAA-3'; SDHD-R:5'-GGTTGATGTTTGTTCCGATT-3'.

4. A kit for detecting the molecular marker according to claim 1, characterized in that: Including the one described in claim 3 Primer pairs.

5. The kit according to claim 4, characterized in that: Also included are DNA extraction reagents and PCR amplification reagents.

6. Use of the molecular marker according to claim 1, the primer pair according to claim 3, or the kit according to claim 4 in detecting the resistance of Panonychus citri to ethidium bromide.

7. A method for detecting molecular markers of citrus mites' resistance to ethidium bromide, characterized in that: First, the total DNA of the citrus panonychus mites to be tested is extracted, and the total DNA is used as a template to perform PCR amplification with the primer pair described in claim 3 to obtain a succinate dehydrogenase SDHD subunit gene fragment containing a mutation region, and the obtained gene fragment is sequenced with the molecular marker of citrus panonychus mites to ethtoxafenib in claim 1 to determine the resistance of the citrus panonychus mites to ethtoxafenib.

8. The detection method according to claim 7, characterized in that: If the gene fragment sequence obtained by PCR amplification is the sequence of the molecular marker according to claim 1, it is determined that the citrus mites to be tested are resistant to ethidium bromide.

9. The detection method according to claim 7, characterized in that: The reaction system of the PCR amplification is: 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 ddH2O is added to 50 μL.

10. The detection method according to claim 9, characterized in that: The PCR amplification program is: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, extension at 72°C for 30 sec, 40 cycles; and finally extension at 72°C for 5 min.

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