Soybean disease and pest resistant molecular marker assisted breeding method

Abstract

The invention discloses a soybean disease-resistant and insect-resistant molecular marker-assisted breeding method. The method comprises the following steps: collecting and storing soybean germplasm resources with different disease-resistant and insect-resistant characters; the method comprises the following steps: screening a molecular marker closely linked with disease and pest resistance characters from a soybean genome by using a genome sequencing method; based on the screened molecular marker, constructing a soybean genetic map, and defining a linkage relationship between the molecular marker and a target disease and pest resistant gene; detecting the genotype of the molecular marker in a soybean material to be bred through a molecular biological method, carrying out auxiliary selection according to the linkage relationship between the molecular marker and the target disease and pest resistant gene, and screening out soybeans carrying the target disease and pest resistant gene; soybean materials which are excellent in genetic background and carry target disease and pest resistant genes are selected as parents to carry out cross combination, and through multi-generation backcross and selfing, the genetic background is further purified, and target characters are stabilized. The method can greatly shorten the breeding period and improve the breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of soybean breeding methods, and in particular to a soybean disease and insect resistance molecular marker assisted breeding method. Background Art

[0002] The scientific name of soybean is max(L)Merrill, and its alias is yellow soybean (including green beans, black beans, purple beans and Giycine beans, etc.). Soybean belongs to the Fabaceae family, the Fabaceae subfamily, the Glycine genus, and the Glycine species. It is an annual herb native to my country. It has been cultivated in my country since ancient times, and has a 5,000-year history of cultivation. It is now widely cultivated throughout the country, and is produced in Northeast China, North China, Shaanxi, Sichuan and the lower reaches of the Yangtze River. It is mostly cultivated in the Yangtze River Basin and the southwest, and the quality of soybeans in Northeast China is the best. Traditional soybean breeding methods rely on field phenotypic screening, which has the disadvantages of long cycles, low efficiency and susceptibility to environmental interference. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to provide a soybean disease and insect resistance molecular marker assisted breeding method which can greatly shorten the breeding cycle and improve the breeding efficiency.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: a soybean disease and insect resistance molecular marker assisted breeding method, comprising the following steps:

[0005] S1: Collect and preserve soybean germplasm resources with different disease and insect resistance traits;

[0006] S2: Using genome sequencing methods, molecular markers that are closely linked to pest and disease resistance traits are screened in the soybean genome;

[0007] S3: Based on the screened molecular markers, a soybean genetic map is constructed to clarify the linkage relationship between the molecular markers and the target pest and disease resistance genes;

[0008] S4: Detect the molecular marker genotypes in the soybean materials to be bred by molecular biological methods, and conduct auxiliary selection based on the linkage relationship between the molecular markers and the target pest and disease resistance genes to screen out soybeans carrying the target pest and disease resistance genes;

[0009] S5: Select soybean materials with excellent genetic background and carrying target pest and disease resistance genes as parents, perform hybridization, and further purify the genetic background and stabilize the target traits through multiple generations of backcrossing and self-pollination;

[0010] S6: Conduct field trials on the selected excellent hybrid soybean combinations, comprehensively evaluate their disease and pest resistance, yield and quality, and ultimately select new soybean varieties that meet the breeding goals.

[0011] The beneficial effects of adopting the above technical solution are: the method described in this application avoids a large amount of field phenotypic screening through molecular marker-assisted selection, significantly shortening the breeding cycle; molecular marker-assisted breeding has the advantages of being fast, accurate, and not affected by environmental conditions, which improves breeding efficiency. And based on the close linkage relationship between molecular markers and target disease and insect resistance genes, accurate breeding of target traits is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0013] Figure 1 is a flow chart of the method described in an embodiment of the present invention. DETAILED DESCRIPTION

[0014] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] like Figure 1 As shown, the embodiment of the present invention discloses a soybean disease and insect resistance molecular marker assisted breeding method, comprising the following steps:

[0016] S1: Collect and preserve soybean germplasm resources with different disease and insect resistance traits;

[0017] S2: Using genome sequencing methods, molecular markers that are closely linked to pest and disease resistance traits are screened in the soybean genome;

[0018] S3: Based on the screened molecular markers, a soybean genetic map is constructed to clarify the linkage relationship between the molecular markers and the target pest and disease resistance genes;

[0019] S4: Detect the molecular marker genotypes in the soybean materials to be bred by molecular biological methods, and conduct auxiliary selection based on the linkage relationship between the molecular markers and the target pest and disease resistance genes to screen out soybeans carrying the target pest and disease resistance genes;

[0020] S5: Select soybean materials with excellent genetic background and carrying target pest and disease resistance genes as parents, perform hybridization, and further purify the genetic background and stabilize the target traits through multiple generations of backcrossing and self-pollination;

[0021] S6: Conduct field trials on the selected excellent hybrid soybean combinations, comprehensively evaluate their disease and pest resistance, yield and quality, and ultimately select new soybean varieties that meet the breeding goals.

[0022] Furthermore, the step S2 specifically includes the following method:

[0023] 1) Select parent materials: First, identify the disease and insect resistance traits that need to be screened, such as the ability to resist a specific disease or pest, and select soybean varieties with obvious differences in disease and insect resistance traits as parents, for example, a highly disease and insect resistant variety and a susceptible variety.

[0024] 2) Construction of genetic population: hybridizing selected parents to produce F1 soybean varieties; as needed, self-pollinating F1 soybean varieties to produce F2 soybean varieties to enrich target traits;

[0025] 3) Genome sequencing: Extract DNA from individuals of parental and F2 generation soybean varieties; use high-throughput sequencing methods to perform whole-genome resequencing of individuals of parental and partial genetic populations to obtain complete genome sequence information for each individual.

[0026] 4) Variant detection and annotation: The sequence obtained by sequencing is compared with the reference genome to detect variant sites such as single nucleotide polymorphism (SNP), insertion / deletion (InDel), structural variation (SV); functional annotation is performed on the detected variant sites to determine which variants are located in the gene region related to disease and pest resistance traits;

[0027] 5) Association analysis and molecular marker development: Analyze the degree of association between the variant site and the pest and disease resistance trait using statistical methods (such as GWAS, QTL analysis, etc.) to find the variant site that is closely linked to the trait; Based on the results of the association analysis, perform molecular markers, which are used for genotype identification in subsequent breeding;

[0028] Furthermore, the step S4 specifically includes the following method:

[0029] 1) Design of specific primers: Based on the selected molecular markers, which can be SSR (simple sequence repeats), SNP (single nucleotide polymorphisms) or other types of DNA markers; design of specific PCR primers, which are used to amplify the DNA fragments including the molecular markers for subsequent genotyping analysis;

[0030] 2) Extracting soybean DNA: extracting genomic DNA from the soybean material to be bred;

[0031] 3) PCR amplification and genotype analysis: PCR amplification of the extracted DNA was performed using the designed specific primers to obtain DNA fragments including molecular markers; then, these fragments were separated and detected by electrophoresis to determine the molecular marker genotypes of different soybean materials;

[0032] 4) Analyze linkage relationship: Analyze the linkage relationship between molecular markers and target pest and disease resistance genes based on the genetic linkage map to determine which molecular markers are closely linked to the target gene and the degree of linkage;

[0033] 5) Auxiliary selection: Utilize the linkage relationship between molecular markers and target pest and disease resistance genes for assisted selection. By detecting the molecular marker genotype of the soybean material to be bred, it is indirectly predicted whether it carries the target pest and disease resistance gene, and the soybean material carrying the target gene is selected as the soybean seed carrying the target pest and disease resistance gene.

[0034] Furthermore, the step S5 specifically includes the following method:

[0035] 1) Hybrid combination: formulate a hybridization plan and manage the parents carefully to ensure that they are in the best growth state during hybridization; complete hybridization through artificial emasculation, bagging and pollination during the flowering period of the parents; sow, manage the field and control pests and diseases for the seeds obtained from hybridization; at the same time, conduct preliminary screening of the hybrid offspring to remove individuals that obviously do not meet the requirements;

[0036] 2) Multi-generation backcrossing and selfing: The target disease-resistant gene is introduced into soybean seeds with a good genetic background through multi-generation backcrossing. During backcrossing, the hybrid offspring carrying the target disease-resistant gene is used as the male or female parent, and the soybean seeds with a good genetic background are used as the female parent (or male parent). Through continuous multi-generation backcrossing operations, the target gene is gradually introduced into the good genetic background;

[0037] After backcrossing is completed, multiple generations of self-pollination are carried out to purify the genetic background and stabilize the target traits. When selfing, individuals carrying the target disease and insect resistance gene and with excellent performance are selected as selfing parents. Through multiple generations of self-pollination operations, the target gene is homozygous and fixed in the offspring.

[0038] Furthermore, the step S6 specifically includes the following method:

[0039] 1) Experimental preparation: draw a detailed field layout map, clearly specify the arrangement order of the test varieties, the number of repetitions, and the setting of the protection rows; screen the test seeds, remove the blighted, diseased, insect-infested, and miscellaneous seeds, and dry the seeds to improve the germination rate and mix or coat the seeds with pesticides to prevent the seeds from being infected with bacteria; calculate the amount of seeds required for each row according to the sowing density, pack them into paper bags, and mark the variety name, repetition number, and area code;

[0040] 2) Sowing and field management: According to the field layout, use a ruler to measure the field length, trail length, protection row length and number of ridges, and mark the sowing area with ground signs; use mechanical sowing or manual sowing to ensure uniform sowing and consistent depth; check for deficiencies and fill in gaps after seedlings emerge to ensure full seedlings, remove excess and weak seedlings, and ensure uniform distribution of plants; break up soil compaction through inter-cultivation operations, and remove field weeds before closing ridges; water and fertilize in time according to the growth needs of soybeans, and mainly use phosphorus and potassium fertilizers during the seedling stage;

[0041] 3) Pest and disease monitoring and control: adopt a combination of prevention and control methods, select disease-resistant varieties, regularly monitor the occurrence of diseases during cultivation, and spray pesticides for prevention and control; for common soybean pests, adopt a combination of agricultural prevention and chemical prevention methods;

[0042] 4) Field investigation and data collection: Observe and record the flowering period, maturity period, lodging resistance and disease resistance of the hybrid combination; take samples at various stages of soybean growth and record the number, size and weight of pods; evaluate the growth and development of soybeans through data analysis and predict the final yield; at the same time, measure the quality of soybean seeds;

[0043] 5) Comprehensive evaluation and variety selection: Based on the results of field investigations and data analysis, comprehensively evaluate the pest and disease resistance, yield and quality performance of each hybrid combination; based on the breeding goals, select the excellent varieties that meet the requirements.

Claims

1. A soybean disease and insect resistance molecular marker assisted breeding method, characterized in that The steps include: S1: Collect and preserve soybean germplasm resources with different disease and insect resistance traits; S2: Using genome sequencing methods, molecular markers that are closely linked to pest and disease resistance traits are screened in the soybean genome; S3: Based on the screened molecular markers, a soybean genetic map is constructed to clarify the linkage relationship between the molecular markers and the target pest and disease resistance genes; S4: Detect the molecular marker genotypes in the soybean materials to be bred by molecular biological methods, and conduct auxiliary selection based on the linkage relationship between the molecular markers and the target pest and disease resistance genes to screen out soybeans carrying the target pest and disease resistance genes; S5: Select soybean materials with excellent genetic background and carrying target pest and disease resistance genes as parents, perform hybridization, and further purify the genetic background and stabilize the target traits through multiple generations of backcrossing and self-pollination; S6: Conduct field trials on the selected excellent hybrid soybean combinations, comprehensively evaluate their disease and pest resistance, yield and quality, and ultimately select new soybean varieties that meet the breeding goals.

2. The method for soybean disease and insect resistance molecular marker-assisted breeding according to claim 1, characterized in that: The step S2 specifically includes the following method: 1) Select parent materials: First, identify the disease and insect resistance traits that need to be screened, and select soybean varieties with obvious differences in disease and insect resistance traits as parents; 2) Construction of genetic population: hybridizing selected parents to produce F1 soybean varieties; as needed, self-pollinating F1 soybean varieties to produce F2 soybean varieties to enrich target traits; 3) Genome sequencing: DNA is extracted from individuals of parental and F2 soybean varieties, and the whole genome of parents and some genetic population individuals is resequenced using high-throughput sequencing methods to obtain complete genome sequence information for each individual; 4) Variant detection and annotation: compare the sequenced sequences with the reference genome, detect the variant sites, perform functional annotation on the detected variant sites, and determine which variants are located in the gene regions related to pest and disease resistance traits; 5) Association analysis and molecular marker development: Statistical methods are used to analyze the degree of association between variant sites and pest and disease resistance traits, and to identify variant sites that are closely linked to the traits; based on the results of the association analysis, molecular markers are used for genotype identification in subsequent breeding.

3. The method for soybean disease and insect resistance molecular marker-assisted breeding according to claim 1, characterized in that: The step S4 specifically includes the following method: 1) Designing specific primers: Designing specific PCR primers based on the selected molecular markers, wherein the primers are used to amplify the DNA fragments including the molecular markers for subsequent genotyping analysis; 2) Extracting soybean DNA: extracting genomic DNA from the soybean material to be bred; 3) PCR amplification and genotype analysis: PCR amplification of the extracted DNA was performed using the designed specific primers to obtain DNA fragments including molecular markers; then, these fragments were separated and detected by electrophoresis to determine the molecular marker genotypes of different soybean materials; 4) Analyze linkage relationship: Analyze the linkage relationship between molecular markers and target pest and disease resistance genes based on the genetic linkage map to determine which molecular markers are closely linked to the target gene and the degree of linkage; 5) Auxiliary selection: Utilize the linkage relationship between molecular markers and target pest and disease resistance genes for assisted selection. By detecting the molecular marker genotype of the soybean material to be bred, it is indirectly predicted whether it carries the target pest and disease resistance gene, and the soybean material carrying the target gene is selected as the soybean seed carrying the target pest and disease resistance gene.

4. The soybean disease and insect resistance molecular marker-assisted breeding method according to claim 1, characterized in that: The step S5 specifically includes the following method: 1) Hybrid combination: formulate a hybridization plan and manage the parents to ensure that they are in the best growth state during hybridization; complete hybridization through artificial emasculation, bagging and pollination during the flowering period of the parents; sow the seeds obtained from hybridization, conduct field management and control pests and diseases, and at the same time, conduct preliminary screening of hybrid offspring to remove individuals that obviously do not meet the requirements; 2) Multi-generation backcrossing and selfing: The target disease-resistant gene is introduced into soybean seeds with a good genetic background through multi-generation backcrossing. During backcrossing, the hybrid offspring carrying the target disease-resistant gene is used as the male parent, and the soybean seeds with a good genetic background are used as the female parent. Through continuous multi-generation backcrossing operations, the target gene is gradually introduced into the good genetic background; After backcrossing is completed, multiple generations of self-pollination are carried out to purify the genetic background and stabilize the target traits. When selfing, individuals carrying the target disease and insect resistance gene and with excellent performance are selected as selfing parents. Through multiple generations of self-pollination operations, the target gene is homozygous and fixed in the offspring.

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