Bombyx mori polygene breeding method against amide pesticide

Abstract

The present application relates to the field of silkworm molecular genetic breeding technology, and particularly relates to a silkworm amide pesticide resistance multi-gene polymerization breeding method; a high tolerance variety radiation 7 is used as a resistance donor, and a high yield and high quality silkworm variety is selected as a receptor parent; 16 detoxification enzyme genes of carboxylesterase, cytochrome P450 and glutathione-S-transferase family are developed into molecular markers and specific primers; the radiation 7 is used as a male parent to cross with the receptor parent to construct an F2 separation population, a plurality of generations of continuous backcrossing is carried out, the genotype is detected by combining molecular markers, the high expression individual of the multiple detoxification enzymes is screened, the resistance directional selection is carried out through the sublethal concentration pesticide stress and the mating rate of male moths, the individual is continuously self-crossed for more than 5 generations, the gene homozygosity is identified by the molecular marker, and the silk production performance is evaluated; and the cross combination is prepared again; the directional polymerization and stable inheritance of the multiple detoxification enzyme resistance genes are realized, and the overall resistance and genetic stability of the silkworm to the amide pesticide are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of silkworm molecular genetic breeding technology, and in particular to a method for multi-gene aggregation breeding of silkworms resistant to amide pesticides. Background Technology

[0003] Currently, most mulberry orchards are adjacent to farmland. After pesticides are sprayed on farmland, pesticide residues can easily contaminate mulberry leaves through airborne drift, dust deposition, and rainwater runoff. Silkworms are oligophagous, monophagous insects that rely solely on mulberry leaves for food. They are extremely sensitive to amide and pyrethroid pesticides. Even trace amounts of pesticide residues can cause chronic or acute poisoning in silkworms, disrupting normal growth and development, interfering with male moths' courtship and mating behavior, leading to decreased cocooning rates, deterioration of cocoon silk quality, and even the death of entire batches of silkworms, seriously affecting the stable production and income of the sericulture industry.

[0004] Currently, conventional silkworm breeding focuses on improving cocoon silk yield and silk quality traits. Traditional pesticide resistance breeding relies solely on the introduction of a single resistance gene. The breeding methods are extensive, the screening methods are simple, and there is a lack of targeted breeding technology that integrates multiple detoxification enzyme genes. As a result, the resistance level is low, the genetic stability is poor, and it is difficult to cope with the compound pollution of multiple amide pesticides in the field. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-gene polymerization breeding method for silkworms resistant to amide pesticides. This method aims to solve the technical problems of existing resistance breeding technologies that rely solely on the introduction of a single resistance gene, have extensive breeding methods and limited screening techniques, lack targeted breeding technology for the synergistic polymerization of multiple detoxification enzyme genes, and result in low resistance levels, poor genetic stability, and difficulty in dealing with the combined pollution of multiple amide pesticides in the field.

[0006] To achieve the above objectives, the present invention employs a multi-gene aggregation breeding method for silkworms resistant to amide pesticides, comprising the following steps: The radiation-mutated high-tolerance variety 'Fu 7' was selected as the resistance donor parent, and conventional silkworm varieties with high yield, high quality, and excellent cocoon silk traits were selected as the recipient parent. Specific molecular markers were developed for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione S-transferase family, and gene amplification-specific primers were designed and synthesized. Artificial hybridization was carried out using Radiation 7 as the male parent and high-yielding, high-quality recipient parent as the female parent to construct a multi-component hybrid F2 detoxification enzyme gene aggregation and segregation population. Using high-yielding and high-quality recipient parents as recurrent parents, multiple generations of continuous backcrossing were carried out. Samples of silkworms were collected from each generation to detect the genotypes of 16 detoxification enzyme genes, and individuals carrying multiple detoxification enzyme high-expression alleles were screened and retained. Targeted breeding was carried out by applying sublethal concentrations of pyrethroid pesticides, and tolerance was assessed in combination with the mating rate of male moths to screen for individuals with superior resistance. Purified individuals were continuously self-pollinated for more than 5 generations, and the homozygosity of the target gene was confirmed by combining molecular markers, and the silk production performance was evaluated; Hybrid combinations were created by combining genetically homozygous resistant strains with high-yield and high-quality silkworm varieties. Regional breeding trials were conducted, and the identification, approval, and promotion of the varieties were completed.

[0007] Among the steps involved in screening the radiation-mutated, highly tolerant variety 'Fu 7' as the resistance donor parent and selecting high-yielding, high-quality conventional silkworm varieties with excellent cocoon and silk traits as the recipient parent: We collected radiation-mutated germplasm materials preserved in the silkworm germplasm resource bank, conducted preliminary tests on tolerance to pyrethroid and amide pesticides, and determined the median lethal concentration (LC50) for each variety. 50 We screened out the radiation-induced mutant variety 'Fu 7', which has significantly higher pesticide tolerance than ordinary silkworms and stable genetic traits, and used it as a parental donor for the resistance gene. From the main silkworm varieties promoted in production, conventional silkworm varieties with strong physique, high survival rate, high cocoon layer rate, excellent silk quality, wide adaptability, but sensitive to pesticides are selected as breeding recipient parents and recurrent backcross parents. Single-moth isolation breeding was carried out on the donor and recipient parents of Radiation 7, and they were raised under standardized environmental conditions; Field and indoor pre-feeding identification confirmed that the pesticide tolerance trait of the donor parent could be stably inherited and that the economic traits of the silk cocoon of the recipient parent were excellent, thus completing the selection and mating of the parents.

[0008] In the steps of developing specific molecular markers for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione S-transferase family, and designing and synthesizing gene amplification-specific primers: The gene compositions of 16 target detoxification enzymes were determined, including 8 carboxylesterase genes, 2 serine protease inhibitor genes, 1 chymotrypsin gene, 3 lipase genes, 1 cytochrome P450 gene, and 1 glutathione S-transferase gene. The full-length nucleotide sequences of 16 detoxification enzyme genes were downloaded from the SilkDB database of silkworms, and the conserved regions and specific variant sites of the genes were compared using bioinformatics software. SNP and InDel molecular markers were developed based on gene-specific sites, and Primer Premier 5.0 software was used to design gene-specific upstream and downstream PCR amplification primers. Primers were synthesized, a real-time PCR reaction system was configured, and a standard procedure for simultaneous genotyping and expression detection of 16 detoxification enzyme genes was established.

[0009] In the step of constructing a multi-generational hybrid F2 detoxification enzyme gene aggregation and segregation population by artificially hybridizing Radi7 as the male parent and a high-yielding and high-quality recipient parent as the female parent: The male moths of the irradiated moths and the female moths of the recipient parent emerged simultaneously. Healthy and vigorous adults were selected for artificial pairing and hybridization. After hybridization, the ants lay eggs, are induced to grow green, and are collected. They are then raised in a standardized manner under uniform temperature, humidity, light, and mulberry leaf feeding conditions until they cocoon, pupate, and emerge as adults, thus obtaining the F1 generation population. Selective self-pollination of F1 generation moths with uniform traits was carried out, and strict isolation and rearing of individual moths in enclosures was implemented. F2 generation segregating populations are obtained by self-pollinating the F1 generation. The population retains a sufficient number of individuals to form a breeding population carrying multiple detoxification enzyme genes that have recombinated and segregated.

[0010] Among them, in the steps of using high-yielding and high-quality recipient parents as recurrent parents for multiple generations of continuous backcrossing, collecting silkworm samples from each generation to test the genotypes of 16 detoxification enzymes, and screening and retaining individuals carrying multiple high-expression alleles of detoxification enzymes: Using superior single moths from the F2 population as non-recurrent parents and high-yielding, high-quality recipient parents as recurrent maternal parents, continuous backcrossing was carried out for multiple generations, with the number of backcross generations controlled between 4 and 6. Each generation of silkworms was raised to the third instar larval stage, and a small number of tissue samples were collected from the tail of the silkworms to extract total genomic DNA and test the purity and concentration of the DNA. Using quantitative real-time PCR technology and developed molecular markers and specific primers, genotyping of 16 detoxification enzyme genes was performed on each generation of samples simultaneously. Based on gene expression levels and genotype results, individuals carrying multiple high-expression alleles of detoxification enzymes are selected for breeding, while individuals with single genes, low expression, or no resistance genes are eliminated, and then proceed to the next round of backcrossing and breeding.

[0011] Among the steps involved in targeted breeding under sublethal concentrations of pyrethroid pesticide stress, assessing tolerance based on male moth mating rates, and screening for individuals with superior resistance: Prepare 2.0–2.5 mg / L sublethal concentration pyrethroid pesticide solutions, and use the mulberry leaf impregnation method to prepare pesticide-treated mulberry leaves; During the second to fourth instar larval stages of silkworms, mulberry leaves containing pesticides were continuously fed to induce continuous pesticide stress, while a clean water control group was set up. After the silkworm moths emerged, the mating behavior and mating rate of male moths were statistically analyzed at 0h, 1h, 2h, 3h, 4h, 5h, and 6h after pesticide treatment, in groups, to evaluate pesticide tolerance from the perspective of reproductive behavior. Based on the overall larval survival status, uniformity of growth and development, presence of poisoning and deformity symptoms, and mating rate of male moths, superior individuals with strong tolerance, normal development, and unrestricted mating ability are selected for breeding.

[0012] Among these steps, the purification and selection of individuals involves continuous self-pollination for more than 5 generations, confirmation of target gene homozygosity using molecular markers, and evaluation of silk production performance. The resistant single moths obtained by pesticide stress screening were continuously self-pollinated for more than 5 generations using a single-moth isolation method; By sampling the three-year-old population of each generation of self-pollinated population, the genotypes of 16 detoxification enzyme genes were detected by molecular marker fluorescence quantitative PCR, and homozygous lines were screened generation by generation. A systematic feeding experiment was conducted on the genetically homozygous strains to determine the economic traits of developmental duration, larval survival rate, cocooning rate, total cocoon weight, cocoon layer ratio, silk length, and purity. Screen for homozygous resistance lines that are completely homozygous for detoxification enzyme genes, have uniform traits, are genetically stable, and have silk production performance at the level of the main recommended varieties.

[0013] Among these steps, the process involves using genetically homozygous resistant strains to create hybrid combinations with high-yield and high-quality silkworm varieties, conducting regional breeding trials, and completing the identification, approval, and promotion of varietal traits. Homozygous resistant Chinese lines were selected and reciprocally crossed with Japanese lines, while multi-varietal hybrid combinations were prepared with high-yielding varieties. Multiple regional trials were set up in different major silkworm producing areas to unify feeding and management standards and compare and measure the survival rate, growth and development traits, cocoon and silk quality, and yield of various hybrid combinations under pesticide stress. The optimal hybridization combination was selected by considering factors such as overall resistance level, feeding adaptability, cocoon yield per 10,000 silkworms, and cocoon silk quality. Complete the testing of variety specificity, uniformity, and stability, as well as regional trials and identification; fulfill the silkworm variety approval process; and carry out large-scale silkworm breeding and widespread application.

[0014] This invention discloses a multi-gene polymerization breeding method for silkworms resistant to amide pesticides, comprising the following steps: screening a radiation-mutated, highly tolerant variety, Radiation 7, as the resistance donor parent, and selecting a high-yielding, high-quality conventional silkworm variety with excellent cocoon silk traits as the recipient parent; developing specific molecular markers for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione-S-transferase family, and designing and synthesizing gene amplification-specific primers; artificially hybridizing Radiation 7 as the male parent and the high-yielding, high-quality recipient parent as the female parent to construct a multi-generational hybrid F2 detoxification enzyme gene polymerization segregating population; and conducting multiple generations of continuous backcrossing using the high-yielding, high-quality recipient parent as the recurrent parent, collecting silkworm samples from each generation to detect the genotypes of the 16 detoxification enzyme genes, and screening for and maintaining... Individuals carrying multiple detoxification enzyme high-expression alleles were retained; targeted breeding was carried out by applying sublethal concentrations of pyrethroid pesticides, and tolerance was assessed by combining male moth mating rates to screen for resistant individuals; purified and selected individuals were continuously self-crossed for more than 5 generations, and the homozygosity of target genes was confirmed by combining molecular markers, and silk production performance was assessed; hybrid combinations were prepared by using homozygous resistant lines with high-yielding and high-quality silkworm varieties, regional breeding trials were carried out, and variety trait identification, approval and promotion were completed; through the above methods, the targeted aggregation and stable inheritance of multiple detoxification enzyme resistance genes were achieved; the limitations of traditional single-gene resistance were effectively overcome, and the overall resistance and genetic stability of silkworms to amide pesticides were significantly improved, enabling them to tolerate multiple amide pesticide compound pollution in the field. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of the steps of the multi-gene polymerization breeding method for silkworms resistant to amide pesticides according to the present invention.

[0017] Figure 2 This is a flowchart of steps S100 of the present invention.

[0018] Figure 3 This is a flowchart of steps S200 of the present invention.

[0019] Figure 4 This is a flowchart of steps S300 of the present invention.

[0020] Figure 5 This is a flowchart of steps S400 of the present invention.

[0021] Figure 6 This is a flowchart of steps S500 of the present invention.

[0022] Figure 7 This is a flowchart of steps S600 of the present invention.

[0023] Figure 8 This is a flowchart of steps S700 of the present invention. Detailed Implementation

[0024] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application.

[0025] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0026] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this application, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0027] Please see Figures 1-8 This invention provides a method for multi-gene aggregation breeding of silkworms resistant to amide pesticides, comprising the following steps: S100: The radiation-mutated high-tolerance variety 'Fu 7' was selected as the resistance donor parent, and conventional silkworm varieties with high yield, high quality, and excellent cocoon silk traits were selected as the recipient parent.

[0028] In this embodiment, the radiation-mutated, highly tolerant variety 'Radiation 7' ​​was selected as the resistance donor parent, and a high-yielding, high-quality conventional silkworm variety with excellent cocoon and silk characteristics was selected as the recipient parent. The specific process is as follows: S101: Collect radiation-induced mutagenesis germplasm materials preserved in the silkworm germplasm resource bank, conduct preliminary tests on tolerance to pyrethroid and amide pesticides, and determine the median lethal concentration (LC50) for each variety. 50 We screened out the radiation-induced mutant variety 'Fu 7', which has significantly higher pesticide tolerance than ordinary silkworms and stable genetic traits, and used it as a parental donor for the resistance gene. S102: Select conventional silkworm varieties that are robust, have high survival rates, high cocoon layer ratios, excellent silk quality, and wide adaptability but are sensitive to pesticides from the main silkworm varieties promoted for production, and use them as breeding recipient parents and recurrent backcross parents; S103: Single-moth isolation breeding of the irradiation 7 donor and recipient parents, and standardized environmental conditions for rearing; S104: Field and indoor pre-feeding identification to confirm that the pesticide tolerance trait of the donor parent can be stably inherited and that the economic traits of the silk cocoon of the recipient parent are excellent, and to complete the selection and mating of the parents.

[0029] In the above process, radiation-mutated germplasm materials preserved in the silkworm germplasm resource bank were collected, and preliminary tests on the tolerance of pyrethroid and amide pesticides were conducted. The LC50 of each variety was determined, and the radiation-mutated variety "Fu 7," which has significantly higher pesticide tolerance than ordinary silkworms and stable genetic traits, was screened out as a parental donor for the resistance gene. From the main silkworm varieties promoted for production, conventional silkworm varieties with strong physique, high survival rate, high cocoon layer ratio, excellent silk quality, and wide adaptability, but sensitive to pesticides, were selected. As breeding recipient parents and recurrent backcross parents; single-moth isolation breeding of Radiation 7 donor parents and recipient parents, and standardized environmental conditions for rearing; field and indoor pre-rearing identification to confirm that the pesticide tolerance trait of the donor parents can be stably inherited and that the cocoon silk economic traits of the recipient parents are excellent, and the parent selection and mating are completed. In the above steps, through the combination of tolerance physiological identification and agronomic trait evaluation, the resistance donor and high-yield recipient parents are accurately determined, providing a homozygous and stable germplasm basis for subsequent multi-gene integration breeding.

[0030] S200: Develop specific molecular markers for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione-S-transferase family, and design and synthesize gene amplification-specific primers.

[0031] In this embodiment, specific molecular markers were developed for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione S-transferase family. Gene amplification-specific primers were designed and synthesized. The specific process is as follows: S201: The gene composition of 16 target detoxification enzymes was determined, including 8 carboxylesterase genes, 2 serine protease inhibitor genes, 1 chymotrypsin gene, 3 lipase genes, 1 cytochrome P450 gene, and 1 glutathione-S-transferase gene. S202: The full-length nucleotide sequences of 16 detoxification enzyme genes were downloaded from the SilkDB database of silkworms, and the conserved regions and specific variant sites of the genes were compared using bioinformatics software; S203: SNP and InDel molecular markers were developed based on gene-specific sites, and Primer Premier 5.0 software was used to design gene-specific upstream and downstream PCR amplification primers. S204: Synthesize primers, configure the real-time PCR reaction system, and establish a standard procedure for simultaneous genotyping and expression detection of 16 detoxification enzyme genes.

[0032] In the above process, the gene composition of 16 target detoxification enzymes was first determined, including 8 carboxylesterase genes, 2 serine protease inhibitor genes, 1 chymotrypsin gene, 3 lipase genes, 1 cytochrome P450 gene, and 1 glutathione-S-transferase gene. Then, the full-length nucleotide sequences of the 16 detoxification enzyme genes were downloaded from the silkworm SilkDB database, and bioinformatics software was used to compare conserved regions and specific variant sites. Based on gene-specific sites, SNP and InDel molecular markers were developed, and gene-specific upstream and downstream PCR amplification primers were designed using PrimerPremier 5.0 software. Subsequently, primers were synthesized, and quantitative real-time PCR (qPCR) was configured. In the above steps, through gene sequence analysis, molecular marker development, and primer optimization and synthesis, a molecular detection system adapted to silkworm detoxification enzyme genes was constructed, providing technical support for subsequent precise genotype screening.

[0033] S300: Using Radiation 7 as the male parent and high-yield, high-quality recipient parent as the female parent, artificial hybridization was carried out to construct a multi-component hybrid F2 detoxification enzyme gene aggregation and segregation population.

[0034] In this embodiment, artificial hybridization was carried out using Radiation 7 as the male parent and a high-yielding, high-quality recipient parent as the female parent to construct a multi-generational hybrid F2 detoxification enzyme gene aggregation and segregation population. The specific process is as follows: S301: Seven male moths awaiting irradiation and female parent moths of the recipient emerged simultaneously. Healthy and vigorous adults were selected for artificial pairing and hybridization. S302: After hybridization, the ants lay eggs, are induced to grow green, and are collected. They are then raised in a standardized manner under uniform temperature, humidity, light, and mulberry leaf feeding conditions until they cocoon, pupate, and emerge as adults, thus obtaining the F1 generation population. S303: Selective self-pollination of F1 generation moths with uniform traits, and strict isolation and rearing of individual moths in enclosures; S304: F2 generation segregating population is obtained by self-pollination of F1 generation offspring. The population retains a sufficient number of individuals to form a breeding population carrying multiple detoxification enzyme genes for recombination and segregation.

[0035] In the above process, male moths of the irradiated species 7 and female parent moths of the recipient emerged simultaneously. Healthy and vigorous adults were selected for artificial pairing and hybridization. After hybridization, they were allowed to lay eggs, develop larvae, and be collected. They were then raised under standardized conditions of uniform temperature, humidity, light, and mulberry leaf feeding until they spun cocoons, pupated, and emerged, thus obtaining the F1 generation population. Then, F1 generation moths with uniform traits were selected for self-pollination and breeding, and strictly isolated in individual enclosures. The F2 generation segregating population was then obtained through self-pollination of the F1 generation. The population retained a sufficient number of individuals to form a breeding population carrying multiple detoxification enzyme genes that were recombined and segregated. In the above steps, single-moth hybridization, single-moth self-pollination, and isolated rearing models were used to construct an F2 population with rich genetic background segregation, realizing the creation of a basic population for the recombination and aggregation of 16 detoxification enzyme resistance genes.

[0036] S400: Using high-yielding and high-quality recipient parents as recurrent parents, multiple generations of continuous backcrossing are carried out. Samples of silkworms are collected in each generation to detect the genotypes of 16 detoxification enzyme genes, and individuals carrying multiple detoxification enzyme high-expression alleles are screened and retained.

[0037] In this embodiment, high-yielding and high-quality recipient parents are used as recurrent parents for multiple generations of continuous backcrossing. Samples of silkworms are collected from each generation to detect the genotypes of 16 detoxification enzymes. Individuals carrying multiple high-expression alleles of detoxification enzymes are screened and retained. The specific process is as follows: S401: Using the superior single moth of the F2 population as the non-recurrent parent and the high-yielding and high-quality recipient parent as the recurrent maternal parent, carry out continuous backcrossing for multiple generations, with the number of backcross generations controlled at 4 to 6. S402: Each generation of silkworms is raised to the third instar larval stage, and a small amount of tissue samples are collected from the tail of the silkworm to extract total genomic DNA and test the purity and concentration of the DNA. S403: Using real-time PCR technology, developed molecular markers and specific primers, genotyping of 16 detoxification enzyme genes was performed on each generation of samples simultaneously. S404: Based on gene expression levels and genotype results, individuals carrying multiple high-expression alleles of detoxification enzymes are selected for breeding, while individuals with single genes, low expression, or no resistance genes are eliminated, and then enter the next round of backcrossing and breeding.

[0038] In the above process, firstly, superior single moths from the F2 population were used as non-recurrent parents, and high-yielding and high-quality recipient parents were used as recurrent maternal parents. Multiple generations of backcrossing were conducted, with the number of backcross generations controlled between 4 and 6. Then, each generation of silkworms was reared to the third instar larval stage, and a small amount of tissue samples were collected from the tail of the silkworms to extract total genomic DNA. The purity and concentration of the DNA were then tested. Using quantitative real-time PCR technology and developed molecular markers and specific primers, genotyping of 16 detoxification enzyme genes was performed on each generation of samples. Next, based on gene expression levels and genotype results, individuals carrying multiple high-expression alleles of detoxification enzymes were selected for breeding, while individuals with single genes, low expression, or no resistance genes were eliminated. These individuals then entered the next round of backcrossing. Through these steps, the high-yielding and high-quality genetic background of the recipient parents was rapidly restored through multiple generations of backcrossing. Combined with molecular marker-assisted genotyping, high-expression alleles of multiple resistance genes were selectively enriched generation by generation, significantly improving breeding selection efficiency.

[0039] S500: Directed breeding is carried out by applying sublethal concentrations of pyrethroid pesticides, and tolerance is assessed in combination with the mating rate of male moths to screen for individuals with superior resistance.

[0040] In this embodiment, sublethal concentrations of pyrethroid pesticides are applied for targeted breeding, and tolerance is assessed in conjunction with the mating rate of male moths to screen for individuals with superior resistance. The specific process is as follows: S501: Prepare a pyrethroid pesticide solution with a sublethal concentration of 2.0–2.5 mg / L, and use the mulberry leaf impregnation method to prepare medicated mulberry leaves; S502: During the second to fourth instar larval stages of silkworms, continuously feed them mulberry leaves containing pesticides to subject them to continuous pesticide stress treatment, and set up a clean water blank control group; S503: After the silkworm moths emerge from their adult form, the mating behavior and mating rate of male moths were statistically analyzed in groups at 0h, 1h, 2h, 3h, 4h, 5h, and 6h after pesticide treatment, to evaluate pesticide tolerance from the perspective of reproductive behavior. S504: Based on the overall larval survival status, uniformity of growth and development, presence of poisoning and deformity symptoms, and mating rate of male moths, select superior individuals with strong tolerance, normal development, and unrestricted mating ability for breeding.

[0041] In the above process, firstly, a sublethal concentration of pyrethroid pesticide solution of 2.0–2.5 mg / L was prepared, and mulberry leaves were prepared using the mulberry leaf soaking method. Then, during the second to fourth instar larval stages of silkworms, the mulberry leaves were continuously fed with the pesticide-treated leaves to conduct continuous pesticide stress treatment, with a blank control group of water set up. After the silkworm moths emerged, the mating behavior and mating rate of male moths were statistically analyzed at 0h, 1h, 2h, 3h, 4h, 5h, and 6h after pesticide treatment to evaluate pesticide tolerance from the perspective of reproductive behavior. Subsequently, based on the larval survival status, uniformity of growth and development, presence or absence of poisoning and deformity symptoms, and mating rate of male moths, superior individuals with strong tolerance, normal development, and no significant inhibition of mating ability were selected for breeding. In the above steps, the sublethal dose of pesticide was used for indoor stress combined with the phenotypic identification of male moth reproductive behavior to selectively screen resistant individuals from the dual dimensions of physiological tolerance and reproductive adaptation, ensuring the stability of the selected varieties in actual field application.

[0042] S600: Purified and selected individuals are continuously self-crossed for more than 5 generations, and the homozygosity of the target gene is confirmed by combining molecular markers, and the silk production performance is evaluated.

[0043] In this embodiment, the purified and selected individuals are continuously self-crossed for more than 5 generations, and the homozygosity of the target gene is confirmed by combining molecular markers, and the silk production performance is evaluated. The specific process is as follows: S601: The resistant single moths obtained by pesticide stress screening are continuously self-pollinated for more than 5 generations using a single-moth isolation method; S602: By sampling the three-year-old population of each generation of self-pollinated population, the genotypes of 16 detoxification enzyme genes were detected by molecular marker fluorescence quantitative PCR, and homozygous lines were screened generation by generation. S603: A systematic feeding experiment was conducted on the homozygous strains to determine the economic traits of developmental duration, larval survival rate, cocooning rate, total cocoon weight, cocoon layer ratio, silk length, and purity. S604: Screening for homozygous resistance lines with completely homozygous detoxification enzyme genes, uniform traits, genetic stability, and silk production performance reaching the level of the main recommended varieties.

[0044] In the above process, firstly, the resistant single moths obtained through pesticide stress screening are continuously self-pollinated for more than 5 generations using a single-moth isolation method. Then, by sampling the third instar of each generation of self-pollinated populations, the genotypes of 16 detoxification enzyme genes are detected using molecular marker-assisted quantitative PCR, and homozygous lines are screened generation by generation. Next, a systematic feeding experiment is conducted on the homozygous lines to determine the economic traits such as developmental period, larval survival rate, cocooning rate, total cocoon weight, cocoon layer ratio, silk length, and purity. Subsequently, resistant homozygous lines with completely homozygous detoxification enzyme genes, uniform traits, genetic stability, and silk production performance reaching the level of the main recommended varieties are screened. In the above steps, genetic homozygosity is achieved through multiple generations of single moth self-pollination. Combined with molecular marker gene identification and systematic evaluation of cocoon and silk traits, homozygous breeding lines with stable resistance and excellent agronomic traits are obtained.

[0045] S700: Using genetically homozygous resistant strains and high-yield, high-quality silkworm varieties to create hybrid combinations, conduct regional breeding trials, and complete variety trait identification, approval, and promotion.

[0046] In this embodiment, a hybrid combination was prepared by combining a genetically homozygous resistant strain with a high-yielding and high-quality silkworm variety, and regional breeding trials were conducted to complete the identification, approval, and promotion of the variety traits. The specific process is as follows: S701: Select homozygous resistant Chinese lines and Japanese lines for reciprocal crosses, and simultaneously create multi-variant hybrid combinations with high-yielding varieties; S702: Set up multi-point regional trials in different major silkworm producing areas, unify feeding and management standards, and compare and measure the survival rate, growth and development traits, cocoon and silk quality, and yield indicators of each hybrid combination under pesticide stress. S703: Screening the optimal hybrid combination based on comprehensive resistance level, feeding adaptability, cocoon yield per 10,000 silkworms and cocoon silk quality; S704: Complete the testing of variety specificity, uniformity, and stability, as well as regional trial identification; fulfill the silkworm variety approval process; and carry out large-scale silkworm breeding and widespread application.

[0047] In the above process, firstly, homozygous resistant Chinese strains and Japanese strains are selected for reciprocal crosses, and simultaneously, multi-varietal hybrid combinations are prepared with high-yielding varieties. Then, multi-point regional trials are set up in different major silkworm producing areas, with unified feeding and management standards, and comparative measurements of pesticide stress survival rate, growth and development traits, cocoon and silk quality, and yield indicators of each hybrid combination. Next, the optimal superior hybrid combination is screened by comprehensively considering resistance level, feeding adaptability, cocoon yield per 10,000 silkworms, and cocoon and silk quality. Finally, variety specificity, uniformity, stability tests and regional trial identification are completed, and the silkworm variety approval process is carried out for large-scale silkworm breeding and large-scale promotion and application. In the above steps, through the crossbreeding of resistant homozygous strains and multi-point regional trials, new hybrid combinations that combine resistance, high yield, and high quality are screened, and the approval and industrial application of new varieties are completed.

[0048] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein.

[0049] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for multi-gene aggregation breeding of silkworms resistant to amide pesticides, characterized in that, Includes the following steps: The radiation-mutated high-tolerance variety 'Fu 7' was selected as the resistance donor parent, and conventional silkworm varieties with high yield, high quality, and excellent cocoon silk traits were selected as the recipient parent. Specific molecular markers were developed for 16 detoxification enzyme genes, including carboxylesterase, cytochrome P450, and glutathione S-transferase family, and gene amplification-specific primers were designed and synthesized. Artificial hybridization was carried out using Radiation 7 as the male parent and high-yielding, high-quality recipient parent as the female parent to construct a multi-component hybrid F2 detoxification enzyme gene aggregation and segregation population. Using high-yielding and high-quality recipient parents as recurrent parents, multiple generations of continuous backcrossing were carried out. Samples of silkworms were collected from each generation to detect the genotypes of 16 detoxification enzyme genes, and individuals carrying multiple detoxification enzyme high-expression alleles were screened and retained. Targeted breeding was carried out by applying sublethal concentrations of pyrethroid pesticides, and tolerance was assessed in combination with the mating rate of male moths to screen for individuals with superior resistance. Purified individuals were continuously self-pollinated for more than 5 generations, and the homozygosity of the target gene was confirmed by combining molecular markers, and the silk production performance was evaluated; Hybrid combinations were developed by combining genetically homozygous resistant strains with high-yield and high-quality silkworm varieties, and regional breeding trials were conducted to complete the identification, approval, and promotion of the varieties.

2. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the steps of screening for radiation-mutated, highly tolerant silkworm variety 'Fu 7' as the resistance donor parent and selecting high-yielding, high-quality conventional silkworm varieties with excellent cocoon and silk traits as the recipient parent: Radiation-induced mutagenic varieties of silkworm were collected from the silkworm germplasm resource bank, and pre-test of pyrethroid and amide pesticide tolerance was carried out. The semi-lethal concentration LC 50 50 of each variety was determined, and the radiation-induced variety 7 with significantly higher pesticide tolerance than ordinary silkworm and stable genetic traits was selected as the resistant gene donor parent. From the main silkworm varieties promoted in production, conventional silkworm varieties with strong physique, high survival rate, high cocoon layer rate, excellent silk quality, wide adaptability, but sensitive to pesticides are selected as breeding recipient parents and recurrent backcross parents. Single-moth isolation breeding was carried out on the donor and recipient parents of Radiation 7, and they were raised under standardized environmental conditions; Field and indoor pre-feeding identification confirmed that the pesticide tolerance trait of the donor parent could be stably inherited and that the economic traits of the silk cocoon of the recipient parent were excellent, thus completing the selection and mating of the parents.

3. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the steps of developing specific molecular markers for 16 detoxification enzyme genes targeting carboxylesterase, cytochrome P450, and the glutathione S-transferase family, and designing and synthesizing gene amplification-specific primers: The gene compositions of 16 target detoxification enzymes were determined, including 8 carboxylesterase genes, 2 serine protease inhibitor genes, 1 chymotrypsin gene, 3 lipase genes, 1 cytochrome P450 gene, and 1 glutathione S-transferase gene. The full-length nucleotide sequences of 16 detoxification enzyme genes were downloaded from the SilkDB database of silkworms, and the conserved regions and specific variant sites of the genes were compared using bioinformatics software. SNP and InDel molecular markers were developed based on gene-specific sites, and Primer Premier 5.0 software was used to design gene-specific upstream and downstream PCR amplification primers. Primers were synthesized, a real-time PCR reaction system was configured, and a standard procedure for simultaneous genotyping and expression detection of 16 detoxification enzyme genes was established.

4. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the steps of constructing a multi-component hybrid F2 detoxification enzyme gene aggregation and segregation population by artificially crossing with Radiation 7 as the male parent and a high-yielding and high-quality recipient parent as the female parent: The male moths of the irradiated moths and the female moths of the recipient parent emerged simultaneously. Healthy and vigorous adults were selected for artificial pairing and hybridization. After hybridization, the ants lay eggs, are induced to grow green, and are collected. They are then raised in a standardized manner under uniform temperature, humidity, light, and mulberry leaf feeding conditions until they cocoon, pupate, and emerge as adults, thus obtaining the F1 generation population. Selective self-pollination of F1 generation moths with uniform traits was carried out, and strict isolation and rearing of individual moths in enclosures was implemented. F2 generation segregating populations are obtained by self-pollinating the F1 generation. The population retains a sufficient number of individuals to form a breeding population carrying multiple detoxification enzyme genes that have recombinated and segregated.

5. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the process of using high-yielding and high-quality recipient parents as recurrent parents for multiple generations of continuous backcrossing, collecting silkworm samples from each generation to test the genotypes of 16 detoxification enzymes, and screening for and retaining individuals carrying multiple high-expression alleles of detoxification enzymes: Using superior single moths from the F2 population as non-recurrent parents and high-yielding, high-quality recipient parents as recurrent maternal parents, continuous backcrossing was carried out for multiple generations, with the number of backcross generations controlled between 4 and 6. Each generation of silkworms was raised to the third instar larval stage, and a small number of tissue samples were collected from the tail of the silkworms to extract total genomic DNA and test the purity and concentration of the DNA. Using quantitative real-time PCR technology and developed molecular markers and specific primers, genotyping of 16 detoxification enzyme genes was performed on each generation of samples simultaneously. Based on gene expression levels and genotype results, individuals carrying multiple high-expression alleles of detoxification enzymes are selected for breeding, while individuals with single genes, low expression, or no resistance genes are eliminated, and then proceed to the next round of backcrossing and breeding.

6. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the process of targeted breeding under sublethal concentrations of pyrethroid pesticide stress, tolerance is assessed by combining male moth mating rates, and superior resistant individuals are selected: Prepare 2.0–2.5 mg / L sublethal concentration pyrethroid pesticide solutions, and use the mulberry leaf impregnation method to prepare pesticide-treated mulberry leaves; During the second to fourth instar larval stages of silkworms, mulberry leaves containing pesticides were continuously fed to induce continuous pesticide stress, while a clean water control group was set up. After the silkworm moths emerged, the mating behavior and mating rate of male moths were statistically analyzed at 0h, 1h, 2h, 3h, 4h, 5h, and 6h after pesticide treatment, in groups, to evaluate pesticide tolerance from the perspective of reproductive behavior. Based on the overall larval survival status, uniformity of growth and development, presence of poisoning and deformity symptoms, and mating rate of male moths, superior individuals with strong tolerance, normal development, and unrestricted mating ability are selected for breeding.

7. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the process of purifying and selecting individuals through continuous self-pollination for more than 5 generations, confirming homozygosity of the target gene using molecular markers, and evaluating silk production performance: The resistant single moths obtained by pesticide stress screening were continuously self-pollinated for more than 5 generations using a single-moth isolation method; By sampling the three-year-old population of each generation of self-pollinated population, the genotypes of 16 detoxification enzyme genes were detected by molecular marker fluorescence quantitative PCR, and homozygous lines were screened generation by generation. A systematic feeding experiment was conducted on the genetically homozygous strains to determine the economic traits of developmental duration, larval survival rate, cocooning rate, total cocoon weight, cocoon layer ratio, silk length, and purity. Screen for homozygous resistance lines that are completely homozygous for detoxification enzyme genes, have uniform traits, are genetically stable, and have silk production performance at the level of the main recommended varieties.

8. The method for multi-gene aggregation breeding of silkworms resistant to amide pesticides as described in claim 1, characterized in that, In the process of using genetically homozygous resistant lines to create hybrid combinations with high-yielding and high-quality silkworm varieties, conducting regional breeding trials, and completing the identification, approval, and promotion of variety traits: Homozygous resistant Chinese lines were selected and reciprocally crossed with Japanese lines, while multi-varietal hybrid combinations were prepared with high-yielding varieties. Multiple regional trials were set up in different major silkworm producing areas to unify feeding and management standards and compare and measure the survival rate, growth and development traits, cocoon and silk quality, and yield of various hybrid combinations under pesticide stress. The optimal hybridization combination was selected by considering factors such as overall resistance level, feeding adaptability, cocoon yield per 10,000 silkworms, and cocoon silk quality. Complete the testing of variety specificity, uniformity, and stability, as well as regional trials and identification; fulfill the silkworm variety approval process; and carry out large-scale silkworm breeding and widespread application.

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