KASP primers for detecting drug resistance mutation of aphis gossypii glover and green peach aphid and application of KASP primers

Abstract

The invention discloses a group of KASP primers for detecting insecticide resistance mutation of aphis gossypii glover and green peach aphid and application of the KASP primers, and relates to the technical field of agricultural biology. The KASP labeled primer group disclosed by the invention comprises a primer combination which is used for detecting resistance mutations of AgoyV62I, AgoyR81T, AgoyK264E, MperR81T, MperV101I, AgoyM918V, AgoyM918LAC, AgoyL1014FCT, MperM918LAT, MperL1014FCT, MperS431F, MperM918TTC, MperM918I, AgoyA302S, AgoyS431F and MperA302G to imidacloprid, deltamethrin, carbofuran, pirimicarb and fipronil. And detecting the drug resistance mutation of the aphis gossypii glover or green peach aphid by using one or more primer combinations. The group of KASP labeled primers is good in specificity and high in sensitivity, and can accurately detect the mutation frequency of the drug resistance of the aphis gossypii glover or green peach aphid to the insecticide.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to rapid molecular detection of melon aphid and peach aphid resistance-related mutations. Background Art

[0002] Aphids are a significant agricultural pest, long causing damage to many crops. Not only do they feed on plant sap, affecting crop reproduction, but many aphid species also transmit plant viral diseases, causing further economic losses. Melon aphids and peach aphids are two important pests of vegetables. Melon aphids, in addition to attacking cucurbits and nightshades, are also a significant pest of cotton, while peach aphids, in addition to attacking nightshades and cruciferous vegetables, are also a significant pest of fruit trees such as peaches. Due to their widespread occurrence and severe damage, both melon aphids and peach aphids require regular control in production. Chemical control is the primary method for aphid control. However, due to long-term use of chemical pesticides, these two aphids have developed a high degree of resistance to commonly used pesticides, making them among the most resistant pests. Conducting pest resistance testing to determine the resistance levels of different species can provide guidance for the rational selection of control agents, improve the effectiveness of chemical control, and reduce chemical pesticide use.

[0003] Traditional methods for detecting insecticide resistance in melon aphids and peach aphids rely primarily on bioassays. While reliable, these methods require live specimens, placing high demands on sample collection, transportation, and husbandry. Furthermore, they are slow and labor-intensive, making them unsuitable for testing multiple populations simultaneously. Methods that determine the presence of resistance mutations by amplifying target genes through PCR, sequencing, and sequence comparison do not require live specimens and can test multiple populations simultaneously, but they still present challenges such as long testing cycles and high costs. KASP (competitive allele-specific PCR) utilizes fluorescent markers to accurately detect SNPs (single nucleotide polymorphisms) in sample DNA. Compared to PCR amplification and sequencing, KASP enables high-throughput, low-cost detection of resistance mutations. However, KASP primers specifically designed for specific insecticide resistance are currently unavailable. Therefore, it is necessary to develop a primer and method for detecting the resistance mutation sites V62I, R81T, K264E on the nicotinic acetylcholine (nAChR) gene of melon aphid, the resistance mutation sites M918V, M918LAC, L1014FCT on the sodium ion channel (kdr) gene, the resistance mutation site A302S on the γ-aminobutyric acid (GABA) gene, and the resistance mutation site S431F on the modified acetylcholinesterase (ace2) gene of melon aphid; KASP primers and methods for modifying the resistance mutation sites R81T and V101I on the choline (nAChR) gene, the resistance mutation sites M918LAT, M918TTC, M918I, L1014FCT on the sodium ion channel (kdr) gene, the resistance mutation site A302 on the γ-aminobutyric acid (GABA) gene, and the resistance mutation site S431F on the acetylcholinesterase (ace2) gene are used to monitor the frequency of insecticide resistance mutations in melon aphids and peach aphids in the field. Summary of the Invention

[0004] The main purpose of the present invention is to achieve high-throughput rapid molecular detection of melon aphid and peach aphid resistance to pesticides.

[0005] To achieve the purpose of the present invention, three sets of KASP primer combinations were designed for detecting drug resistance-related SNP variant sites on the nicotinic acetylcholine (nAChR) gene of cucurbit aphids; three sets of KASP primer combinations were designed for detecting drug resistance-related SNP variant sites on the sodium ion channel (kdr) gene of cucurbit aphids; one set of KASP primer combinations was designed for detecting drug resistance-related SNP variant sites on the gamma-aminobutyric acid (GABA) gene of cucurbit aphids; and one set of KASP primer combinations was designed for detecting drug resistance-related SNP variant sites on the acetylcholinesterase (ace2) gene of cucurbit aphids. SP primer combination; two KASP primer combinations for detecting SNP variants associated with insecticide resistance in the nicotinic acetylcholine (nAChR) gene of green peach aphid; four KASP primer combinations for detecting SNP variants associated with insecticide resistance in the sodium ion channel (kdr) gene of green peach aphid; one KASP primer combination for detecting SNP variants associated with insecticide resistance in the γ-aminobutyric acid (GABA) gene of cucurbit aphid; and one KASPy primer combination for detecting SNP variants associated with insecticide resistance in the modified acetylcholinesterase (ace2) gene of green peach aphid;

[0006] Furthermore, the detection primers include a primer combination for detecting the 62nd mutation Agoy_V62I, the 81st mutation Agoy_R81T, and the 264th mutation Agoy_K264E of the nicotinic acetylcholine (nAChR) protein sequence of melon aphid; a primer combination for detecting the 918th mutation Agoy_M918V, Agoy_M918LAC and the 1014th mutation Agoy_L1014FCT of the sodium ion channel (kdr) protein of melon aphid; a primer combination for detecting the 302nd mutation Agoy_A302G of the gamma-aminobutyric acid receptor (GABA) of melon aphid; a primer combination for detecting the 431st mutation Ag of the modified acetylcholinesterase (ACE2) of melon aphid. Primer combination for detecting the 81st mutation Mper_R81T and the 101st mutation Mper_V101I in the nicotinic acetylcholine (nAChR) protein sequence of green peach aphid; primer combination for detecting the 918th mutation Mper_M918TTC, MperM918TTC and Mper_M918I, and the 1014th mutation Mper_L1014FCT in the sodium ion channel (kdr) protein of green peach aphid; primer combination for detecting the 302nd mutation Mper_A302G in the γ-aminobutyric acid receptor (GABA) of green peach aphid; primer combination for detecting the 431st mutation Mper_S431F in the modified acetylcholinesterase (ace2) of green peach aphid;

[0007] Furthermore, the sequences of the two upstream primers included in the primer combination Agoy_V62I are shown in SEQ ID NO.: 1 and SEQ ID NO.: 2, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 3; the sequences of the two upstream primers included in the primer combination Agoy_R81T are shown in SEQ ID NO.: 4 and SEQ ID NO.: 5, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 6; the sequences of the two upstream primers included in the primer combination Agoy_K264E are shown in SEQ ID NO.: 7 and SEQ ID NO.: 8, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 9; the sequences of the two upstream primers included in the primer combination Mper_R81T are shown in SEQ ID NO.: 10 and SEQ ID NO.: 11, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 12 : as shown; the sequences of the two upstream primers included in the primer combination Mper_V101I are shown in SEQ ID NO.: 13 and SEQ ID NO.: 14, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 15; the sequences of the two upstream primers included in the primer combination Agoy_M918V are shown in SEQ ID NO.: 16 and SEQ ID NO.: 17, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 18; the sequences of the two upstream primers included in the primer combination Agoy_M918LAC are shown in SEQ ID NO.: 19 and SEQ ID NO.: 20, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 21; the sequences of the two upstream primers included in the primer combination Agoy_L1014FCT are shown in SEQ ID NO.: 22 and SEQ ID NO.: 23, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 24 The sequences of the two upstream primers contained in the primer combination Mper_M918LAT are shown in SEQ ID NO.: 25 and SEQ ID NO.: 26, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 27; the sequences of the two upstream primers contained in the primer combination Mper_L1014FCT are shown in SEQ ID NO.: 28 and SEQ ID NO.: 29, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 30; the sequences of the two upstream primers contained in the primer combination Mper_S431F are shown in SEQ ID NO.: 31 and SEQ ID NO.: 32, and the sequence of the downstream primer is shown in SEQ ID NO.: 33; the sequences of the two upstream primers included in the primer combination Mper_M918TTC are shown in SEQ ID NO.: 34 and SEQ ID NO.: 35, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 36; the sequences of the two upstream primers included in the primer combination Mper_M918I are shown in SEQ ID NO.: 37 and SEQ ID NO.: 38, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 39; the sequences of the two upstream primers included in the primer combination Agoy_A302S are shown in SEQ ID NO.: 40 and SEQ ID NO.: 41, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 42; the sequences of the two upstream primers included in the primer combination Agoy_S431F are shown in SEQ ID NO.: 43 and SEQ ID NO.: 44, the sequence of the downstream primer is shown in SEQ ID NO.: 45; the sequences of the two upstream primers included in the primer combination Mper_A302G are shown in SEQ ID NO.: 46 and SEQ ID NO.: 47, respectively, and the sequence of the downstream primer is shown in SEQ ID NO.: 48;

[0008] Furthermore, the sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modified tags, which are used to distinguish different alleles during KASP typing;

[0009] The present invention also establishes a high-throughput rapid molecular detection method for insecticide resistance of melon aphids and green peach aphids, and the specific steps are as follows:

[0010] (1) KASP primer combination Agoy_V62I was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was counted to infer the resistance level of the melon aphid population to the insecticide imidacloprid.

[0011] (2) KASP primer combination Agoy_R81T was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as C:C, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:C, it was a heterozygous individual. The frequency of the resistance allele C in the tested population was counted, and the resistance level of the melon aphid population to the insecticide imidacloprid was inferred.

[0012] (3) KASP primer combination Agoy_K264E was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted to infer the resistance level of the melon aphid population to the insecticide imidacloprid.

[0013] (4) KASP primer combination Mper_R81T was used to perform KASP reaction and SNP typing using the DNA of peach aphid as a template. If the fluorescence signal data of the amplified product was typed as C:C, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:C, it was a heterozygous individual. The frequency of the resistance allele C in the tested population was counted, and the resistance level of the peach aphid population to the insecticide imidacloprid was inferred.

[0014] (5) KASP primer combination Mper_V101I was used to perform KASP reaction and SNP typing using the DNA of peach aphid as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was counted, and the resistance level of the peach aphid population to the insecticide imidacloprid was inferred.

[0015] (6) KASP primer combination Agoy_M918V was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted to infer the resistance level of the melon aphid population to the insecticide cypermethrin.

[0016] (7) KASP primer combination Agoy_M918LAC was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as C:C, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as A:C, it was a heterozygous individual. The frequency of the resistance allele C in the tested population was statistically analyzed to infer the resistance level of the melon aphid population to the insecticide cypermethrin.

[0017] (8) KASP primer combination Agoy_L1014FCT was used to perform KASP reaction and SNP typing using the melon aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the melon aphid population to the insecticide cypermethrin was inferred.

[0018] (9) The KASP primer combination Mper_M918LAT was used to perform KASP reaction and SNP typing using the DNA of peach aphid as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as A:A, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:A, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the peach aphid population to the insecticide cypermethrin was inferred.

[0019] (10) The KASP primer combination Mper_L1014FCT was used to perform KASP reaction and SNP typing using the green peach aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the green peach aphid population to the insecticide cypermethrin was inferred.

[0020] (11) The KASP primer combination Mper_S431F was used to perform KASP reaction and SNP typing using the DNA of peach aphid as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the peach aphid population to the insecticide carbofuran.

[0021] (12) The KASP primer combination Mper_M918TTC was used to perform KASP reaction and SNP typing using the green peach aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as C:C, it was a homozygous resistant individual; if the fluorescence signal data was typed as T:T, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual. The frequency of the resistance allele C in the tested population was counted, and the resistance level of the green peach aphid population to the insecticide carbofuran was inferred.

[0022] (13) The KASP primer combination Mper_M918I was used to perform KASP reaction and SNP typing using the green peach aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as A:A, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:A, it was a heterozygous individual. The frequency of the resistance allele A in the tested population was counted, and the resistance level of the green peach aphid population to the insecticide carbofuran was inferred.

[0023] (14) KASP primer combination Agoy_A302S was used to perform KASP reaction and SNP typing using the DNA of melon aphid as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as G:G, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:T, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was counted, and the resistance level of the melon aphid population to the insecticide cypermethrin was inferred.

[0024] (15) KASP primer combination Agoy_S431F was used to perform KASP reaction and SNP typing using DNA of Tetranychus urticae as a template. If the fluorescence signal data of the amplified product was typed as T:T, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as T:C, it was a heterozygous individual. The frequency of the resistance allele T in the tested population was statistically analyzed to infer the resistance level of the melon aphid population to the insecticide cypermethrin.

[0025] (16) The KASP primer combination Mper_A302G was used to perform KASP reaction and SNP typing using the green peach aphid DNA as a template. If the fluorescence signal data of the amplified product was typed as G:G, it was a homozygous resistant individual; if the fluorescence signal data was typed as C:C, it was a homozygous sensitive individual; if the fluorescence signal data was typed as G:C, it was a heterozygous individual. The frequency of the resistance allele G in the tested population was counted, and the resistance level of the green peach aphid population to the insecticide fipronil was inferred.

[0026] The present invention has the beneficial effect of designing a KASP detection primer set for identifying insecticide-resistant individuals, addressing the urgent need for rapid detection of insecticide resistance in melon aphids and green peach aphids in production. This primer set enables rapid, accurate, low-cost, and high-throughput detection of the frequency of insecticide-resistant mutations in melon aphids and green peach aphids. This has broad application prospects in the rational and precise selection of pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 KASP genotype diagram for detecting resistance mutations in melon aphid and peach aphid. DETAILED DESCRIPTION

[0028] The following describes in detail the method for detecting multiple insecticide resistance of melon aphid and green peach aphid using KASP primers designed according to the present invention with reference to specific examples. This detailed description should not be construed as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] Example 1 Screening of KASP Primers for Mutation Sites Related to Pesticide Resistance in Melon Aphid and Green Peach Aphid

[0030] Genomic DNA sequences within 500 base pairs (bp) of the 16 resistance mutation sites in melon aphids and peach aphids were extracted. KASP primers were designed using Primer 3. KASP reactions and SNP typing were performed using DNA from melon aphids and peach aphids as templates. Each KASP primer set was tested for clear typing and a success rate greater than 90%. For each mutation site, a preferred combination was identified, as shown in Table 1.

[0031] Table 1 Preferred KASP primer sequence combinations obtained by screening

[0032]

[0033] Example 2 Application of KASP Primers in Detecting the Frequency of Resistance Mutations in Melon Aphids and Green Peach Aphids

[0034] (1) Sample collection

[0035] Melon aphid samples were collected in 2021 from a field population in a greenhouse at an agricultural cooperative in Daxing District, Beijing (population A) and a field population in a plantation in Daxing District, Beijing (population B). Both populations were host cucumbers. Myzus persicae (Population A) were collected from a field population at a farm in Chaoyang District, Beijing, in 2022. Their host was pepper. Myzus persicae (Population B) were collected from a field population in a greenhouse at an agricultural company in Daxing District, Beijing, in 2021. Their host was rapeseed. Live insects were collected, soaked in 100% alcohol, and stored at -20°C.

[0036] (2) DNA extraction

[0037] Genomic DNA was extracted from 46 individuals of different populations of melon aphid and peach aphid using the DNA lysis crude extraction method.

[0038] (3) KASP amplification, typing, and drug resistance prediction

[0039] The reagents used were 2× KASP master mixture (LGC). A 3 μL reaction system included 1.5 ng of dry powdered genomic DNA, 0.14 μL of primer mix (upstream primer concentration: 12 μmol / L, downstream primer concentration: 30 μmol / L), and 3 μL of 2× KASP master mixture.

[0040] The touchdown reaction procedure included: initial denaturation at 94°C for 15 min; followed by 10 cycles of denaturation at 94°C for 20 s and annealing at 61–55°C for 1 min, with a decrease of 0.6°C per cycle; and 40 cycles of denaturation at 94°C for 20 s and annealing at 55°C for 1 min.

[0041] Finally, check the typing status on the typing detector. Figure 1 The statistical genotype frequencies are shown in Table 2. The statistical allele frequencies are shown in Table 3.

[0042] Table 2 Genotype frequencies at resistance mutation sites in two melon aphid populations and two peach aphid populations

[0043]

[0044] Table 3 Allele frequencies at insecticide resistance mutation sites in two melon aphid populations and two peach aphid populations

[0045]

Claims

1. A KASP primer combination for detecting insecticide-resistant mutations in melon aphids and peach aphids, characterized in that KASP primers containing 5 mutation sites of melon aphid and peach aphid resistance to imidacloprid; KASP primers used to detect 5 mutation sites of melon aphid and peach aphid resistance to deltamethrin; KASP primers used to detect 3 mutation sites of peach aphid resistance to chlorpyrifos; KASP primers used to detect 2 mutation sites of melon aphid resistance to amifenac; KASP primers used to detect 1 mutation site of peach aphid resistance to fipronil.

2. The KASP primers for detecting five mutation sites of melon aphid and peach aphid resistance to imidacloprid according to claim 1, characterized in that A primer combination comprising Agoy_V62I, Agoy_R81T, Agoy_K264E, Mper_R81T and Mper_V101I; a KASP primer for detecting five mutation sites of melon aphid and peach aphid resistance to deltamethrin, characterized by comprising a primer combination of Agoy_M918V, Agoy_M918LAC, Agoy_L1014FCT, Mper_M918LAT and Mper_L1014FCT; a primer for detecting peach aphid resistance to cypermethrin KASP primers for detecting three mutation sites of Budweiser resistance, characterized by including a primer combination of Mper_S431F, Mper_M918TTC and Mper_M918I; a KASP primer combination for detecting two mutation sites of melon aphid resistance to aphid, characterized by including a primer combination of Agoy_A302G and Agoy_S431F; a KASP primer for detecting one mutation site of green peach aphid resistance to fipronil, characterized by including a primer combination of Mper_A302G.

3. The sequences of the two upstream primers and the one downstream primer comprised in the primer combination for detecting Agoy_V62I according to claim 2 are shown in SEQ ID NOs.: 1-3, respectively; the sequences of the two upstream primers and the one downstream primer comprised in the primer combination for detecting Agoy_R81T are shown in SEQ ID NOs.: 4-6, respectively; the sequences of the two upstream primers and the one downstream primer comprised in the primer combination for detecting Agoy_K264E are shown in SEQ ID NOs.: 7-9, respectively; the sequences of the two upstream primers and the one downstream primer comprised in the primer combination for detecting Mper_R81T are shown in SEQ ID NOs.: 10-12, respectively; the sequences of the two upstream primers and the one downstream primer comprised in the primer combination for detecting Mper_V101I are shown in SEQ ID NOs.: 13- 15; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Agoy_M918V are shown in SEQ ID NOs.: 16-18, respectively. The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Agoy_M918LAC are shown in SEQ ID NOs.: 19-21, respectively. The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Agoy_L1014FCT are shown in SEQ ID NOs.: 22-24, respectively. The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Mper_M918LAT are shown in SEQ ID NOs.: 25-27, respectively. The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Mper_L1014FCT are shown in SEQ ID NOs.: 28-30, respectively.The sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Mper_S431F are shown in SEQ ID NOs.: 31-33, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Mper_M918TTC are shown in SEQ ID NOs.: 34-36, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Mper_M918I are shown in SEQ ID NOs.: 37-39, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Agoy_A302S are shown in SEQ ID NOs.: 40-42, respectively; the sequences of the two upstream primers and one downstream primer included in the primer combination for detecting Agoy_S431F are shown in SEQ ID NOs.: 43-45, respectively. The sequences of the two upstream primers and one downstream primer contained in the primer combination for detecting Mper_A302G are shown in SEQ ID NOs.: 46-48, respectively.

4. The KASP primer combination of 16 resistance mutation sites according to claim 2, characterized in that: The sequences of the two upstream primers of each primer combination are respectively connected to different fluorescent modification tags.

5. A method for detecting insecticide-resistant mutations in melon aphids and peach aphids, characterized in that: The KASP primer combination according to claim 2 is used to detect whether there is a drug resistance-related mutation using the DNA of melon aphid or peach aphid as a template.

6. The method for detecting insecticide-resistant mutations of melon aphid and green peach aphid according to claim 5, characterized in that: If the KASP primer combination Agoy_V62I is used, the sample with SNP genotype A:A is a homozygous individual for resistance, the sample with genotype G:G is a homozygous individual for sensitivity, and the sample with genotype G:A is a heterozygous individual; if the KASP primer combination Agoy_R81T is used, the sample with SNP genotype C:C is a homozygous individual for resistance, the sample with genotype G:G is a homozygous individual for sensitivity, and the sample with genotype G:C is a heterozygous individual; if the KASP primer combination Agoy_K264E is used, the sample with SNP genotype G:G is a homozygous individual for resistance Individuals with genotype A:A are sensitive homozygous individuals, and those with genotype G:A are heterozygous individuals; if the KASP primer combination Mper_R81T is used, samples with SNP genotype C:C are resistant homozygous individuals, those with genotype G:G are sensitive homozygous individuals, and those with genotype G:C are heterozygous individuals; if the KASP primer combination Mper_V101I is used, samples with SNP genotype A:A are resistant homozygous individuals, those with genotype G:G are sensitive homozygous individuals, and those with genotype G:A are heterozygous individuals; if the KASP primer combination Mper_V101I is used, samples with SNP genotype A:A are resistant homozygous individuals, those with genotype G:G are sensitive homozygous individuals, and those with genotype G:A are heterozygous individuals; If the primer combination Agoy_M918V is used, the samples with SNP genotype of G:G are homozygous individuals for resistance, those with genotype of A:A are homozygous individuals for sensitivity, and those with genotype of G:A are heterozygous individuals; if the KASP primer combination Agoy_M918LAC is used, the samples with SNP genotype of C:C are homozygous individuals for resistance, those with genotype of A:A are homozygous individuals for sensitivity, and those with genotype of C:A are heterozygous individuals; if the KASP primer combination Agoy_L1014FCT is used, the samples with SNP genotype of T:T are homozygous individuals for resistance. If the KASP primer combination Mper_M918LAT is used, the sample with the SNP genotype of T:T is the homozygous individual for resistance, the sample with the genotype of A:A is the homozygous individual for sensitivity, and the sample with the genotype of T:A is the heterozygous individual; if the KASP primer combination Mper_L1014FCT is used, the sample with the SNP genotype of T:T is the homozygous individual for resistance, the sample with the genotype of C:C is the homozygous individual for sensitivity, and the sample with the genotype of T:C is the heterozygous individual. If the KASP primer combination Mper_S431F is used, the sample with the SNP genotype of T:T is a homozygous individual for resistance, the sample with the genotype of C:C is a homozygous individual for sensitivity, and the sample with the genotype of T:C is a heterozygous individual; if the KASP primer combination Mper_M918TTC is used, the sample with the SNP genotype of C:C is a homozygous individual for resistance, the sample with the genotype of T:T is a homozygous individual for sensitivity, and the sample with the genotype of T:C is a heterozygous individual.If the KASP primer combination Mper_M918I is used, samples with the SNP genotype A:A are homozygous for resistance, those with the genotype G:G are homozygous for sensitivity, and those with the genotype G:A are heterozygous. If the KASP primer combination Agoy_A302S is used, samples with the SNP genotype T:T are homozygous for resistance, those with the genotype G:G are homozygous for sensitivity, and those with the genotype G:T are heterozygous. If the KASP primer combination Agoy_S431F is used, samples with the SNP genotype T:T are homozygous for resistance, those with the genotype C:C are homozygous for sensitivity, and those with the genotype T:C are heterozygous. If the KASP primer combination Mper_A302G is used, samples with the SNP genotype G:G are homozygous for resistance, those with the genotype C:C are homozygous for sensitivity, and those with the genotype G:C are heterozygous.

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