Method for assisting efficient breeding of fresh corn by using molecular marker

Abstract

The invention discloses a molecular marker-assisted efficient breeding method for fresh corn, which comprises the following steps: S1, target character confirmation: determining a target character needing to be improved, and positioning a gene or quantitative character locus (QTL) related to the target character through linkage analysis or whole genome association research (GWAS); the method is reasonable in design, existence and transmission of target genes can be accurately identified, the selection accuracy is improved, target character genotypes can be screened in seedling stages or early generations through molecular marker-assisted selection, the breeding cycle is shortened, and good varieties can be more quickly derived. By means of molecular markers, genotype combination and target character expression of filial generations can be known in advance, a breeding scheme is reasonably planned, the probability of obtaining good varieties is improved, multiple target characters can be selected at the same time, individuals with target genotypes can be rapidly screened out from a large number of groups, and the breeding efficiency is improved. The workload of field trials and phenotype identification is reduced, and breeders can concentrate resources to cultivate good varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted breeding, and in particular to a method for efficiently breeding fresh-edible corn using molecular markers. Background Art

[0002] Molecular marker-assisted breeding is a modern breeding technology. Its basic principle is to use the close linkage relationship between molecular markers and target trait genes to indirectly select individuals with target traits by detecting molecular markers. Molecular markers can be variations in DNA sequences, such as single nucleotide polymorphisms (SNPs) or simple sequence repeats (SSRs). These markers are numerous in the genome and are mostly co-dominant and are not affected by environmental conditions.

[0003] Molecular marker-assisted breeding has the advantages of being fast, accurate, and not affected by environmental conditions. It can significantly improve breeding efficiency, shorten the breeding period, and overcome the difficulties in traditional breeding methods. Unlike transgenic breeding, molecular marker-assisted breeding does not involve direct gene modification, but improves varieties through marker-assisted selection, so it does not involve the safety issues of transgenics.

[0004] Fresh corn is an annual herbaceous plant of the genus Zea in the family Poaceae. Fresh corn is an annual monoecious, cross-pollinated plant with tall plants and strong stems. It is an important food crop and feed crop, and is also the crop with the highest total output in the world. Its planting area and total output are second only to rice and wheat. Fresh corn has always been hailed as a longevity food. It is rich in protein, fat, vitamins, trace elements, cellulose, etc., and has great potential for the development of high-nutrition, high-biological functional foods. However, due to its complex genetics and rich variation, the conventional breeding process has the disadvantages of too long a cycle, too large a coefficient of variation, and effects on the growth and development of offspring. For this reason, we proposed a method for efficient breeding of fresh corn using molecular markers to assist in solving the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art, and to propose a method for efficient breeding of fresh corn using molecular markers to assist, which can accurately identify the existence and transmission of target genes, improve the accuracy of selection, and molecular marker-assisted selection can screen the target trait genotype in the seedling stage or early generations, shorten the breeding cycle, and launch superior varieties more quickly. With the help of molecular markers, the genotype combination and target trait performance of hybrid offspring can be understood in advance, the breeding plan can be reasonably planned, the probability of obtaining superior varieties can be increased, and multiple target traits can be selected at the same time, and individuals with target genotypes can be quickly screened out from a large number of populations, reducing the workload of field trials and phenotypic identification, allowing breeders to concentrate resources on cultivating superior varieties.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for efficiently breeding fresh corn using molecular markers, comprising the following steps:

[0008] S1, target trait confirmation: determine the target traits that need to be improved, and locate the genes or quantitative trait loci (QTLs) associated with the target traits through linkage analysis or genome-wide association studies (GWAS);

[0009] S2, development of molecular markers: development of tightly linked molecular markers for located genes or QTLs;

[0010] S3. Screening of breeding materials: Use the developed molecular markers to screen breeding materials, identify donor parents containing target genes and recipient parents with excellent comprehensive traits, and molecularly mark the target genes in the parents;

[0011] S4, hybridizing the parents to obtain F1 hybrids: artificially hybridizing the selected parents, harvesting F1 seeds, planting them, and observing the phenotypic performance of the target traits;

[0012] S5. Molecular marker detection: Collect leaf tissue from F1 generation plants, extract genomic DNA, and then use selected molecular markers to perform genotype detection on F1 generation individuals to determine whether they contain the target gene and screen out heterozygous individuals containing the target gene;

[0013] S6. Backcrossing or self-pollination: The selected F1 generation heterozygous individuals are self-pollinated or backcrossed with the recipient parent to obtain F2 generation or BC1 generation seeds. After planting, DNA is extracted to detect molecular markers. Based on the test results, individuals containing multiple target genes and with a high background recovery rate are selected;

[0014] S7. Multi-generation selection and identification: Repeat the process of backcrossing or self-pollination and molecular marker detection. After multiple generations of selection, the homozygosity of the target gene and the aggregation of excellent genes are gradually increased, and the combination of these beneficial genes in the breeding material is fixed. At the same time, combined with field phenotypic identification, stable strains with excellent agronomic traits and homozygous target genes are screened out.

[0015] Preferably, in S5, a sampler is selected to collect leaf tissue from the F1 generation plants, the sample is ground using a grinder, 50 μl of NaOH solution is added to the ground sample, and the sample is bathed in 98°C-100°C water for 10 minutes. The sample is taken out of the boiling water, and 50 μl of TE solution is added to neutralize the alkalinity in step S3. This mixed solution contains sample DNA for genotype detection.

[0016] Preferably, in S3, when selecting parents, factors such as the relationship and combining ability between the parents are comprehensively considered to obtain good hybrid vigor.

[0017] Preferably, in S2, the type of molecular marker is SSR marker.

[0018] Preferably, in S4, the parents are planted in a greenhouse, the donor parent A and the recipient parent B are planted twice, and the temperature is adjusted so that their flowering periods coincide. When the parents are crossed, pollen from the male parent is collected and inoculated on the female ear of the female parent, and then bagged for isolation.

[0019] Preferably, in S4, phenotypic selection focuses on plant height, number of ears, fruitfulness and disease resistance level, and molecular markers are combined with phenotypes for selection. Phenotypic selection uses disease resistance and yield as important selection indicators, and further selects and eliminates the inferior based on the results of molecular testing to obtain high-quality plants that meet the breeding goals.

[0020] Preferably, in S1, there may be multiple target genes.

[0021] Preferably, in S4, conventional management is performed on the F1 generation seeds during their growth period, such as fertilizing, watering, and pest and disease control.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Traditional breeding based on phenotypic selection is easily affected by environmental factors, while molecular markers directly detect DNA and are not affected by the environment and developmental stage. They can accurately identify the existence and transmission of target genes and improve the accuracy of selection. Molecular marker-assisted selection can screen the genotype of target traits in the seedling stage or early generations without waiting for the plants to mature and show phenotypes, shortening the breeding cycle and launching excellent varieties more quickly.

[0024] 2. With the help of molecular markers, we can understand the genotype combination and target trait performance of hybrid offspring in advance, plan breeding programs rationally, and increase the probability of obtaining excellent varieties, such as predicting hybrid vigor and selecting advantageous combinations for hybrid breeding. We can also select multiple target traits at the same time, and quickly screen out individuals with target genotypes from a large population, reducing the workload of field trials and phenotypic identification, allowing breeders to concentrate resources on cultivating excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention provides a flow chart of a method for efficiently breeding fresh-edible corn using molecular markers. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below 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, rather than all the embodiments.

[0027] Reference Figure 1 , a method for efficient breeding of fresh-eating corn using molecular markers, comprising the following steps:

[0028] S1. Target trait confirmation: Determine the target trait that needs to be improved, and locate the gene or quantitative trait locus (QTL) related to the target trait through linkage analysis or genome-wide association study (GWAS). There can be multiple target genes. In this application, high resistance to pests and diseases and high yield are selected as target genes.

[0029] S2, Development of molecular markers: Develop tightly linked molecular markers for the located genes or QTLs.

[0030] S3. Screening of breeding materials: using the developed molecular markers to screen the breeding materials, identifying the donor parent containing the target gene and the recipient parent with excellent comprehensive traits, and molecularly marking the target gene in the parent. When selecting the parent, the factors such as the relationship and combining ability between the parents are comprehensively considered to obtain good hybrid advantages. In this application, a fresh-eating corn variety with high resistance to pests and diseases but average yield is selected as the donor parent A, and a variety with high yield but weak resistance to pests and diseases is selected as the recipient parent B. Among them, the fresh-eating corn variety with high resistance to pests and diseases but average yield is selected as the donor parent A, and the variety with high yield but weak resistance to pests and diseases is selected as the recipient parent B. This is only a specific embodiment in this application. The target gene can also be other traits such as lodging resistance, drought resistance, plant type, and luxuriance.

[0031] In this application, the type of molecular marker is SSR marker, and the specific steps are as follows:

[0032] Collect parental biological samples containing target DNA and prepare the required experimental reagents and instruments.

[0033] 1. DNA extraction and quality testing

[0034] 1.1 DNA extraction: Use appropriate methods to extract DNA from samples, such as the CTAB method, SDS method or DNA extraction kit, to obtain pure and complete DNA for subsequent experiments.

[0035] 1.2 Quality testing: Use a UV spectrophotometer to measure the absorbance of DNA at 260nm and 280nm, calculate the OD260 / OD280 ratio, evaluate the purity of DNA, and detect the concentration and integrity of DNA to ensure that it meets the requirements of subsequent experiments.

[0036] 2. Primer Design and Synthesis

[0037] 2.1 Primer design: Design specific primers for the selected molecular marker type and target gene or sequence. Professional software can be used for design to ensure that the specificity of the primers, annealing temperature and other parameters are appropriate.

[0038] 2.2 Synthetic primers: Submit the designed primer sequences to a professional biological company for synthesis.

[0039] 3. PCR Amplification

[0040] 3.1 Prepare the reaction system: Add appropriate amounts of DNA template, primers, dNTPs, Taq enzyme, PCR buffer, etc. to the PCR tube in sequence, adjust the amount of each component according to the different primer and template concentrations, and finally add sterile water to make up the reaction system to the required volume.

[0041] 3.2 Set the amplification program: Place the PCR tube into the PCR instrument and set the appropriate amplification program, which generally includes pre-denaturation, denaturation, annealing, and extension steps. Different molecular markers and primers have different amplification programs. For example, the amplification program for SSR markers is usually 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds, for a total of 30 cycles, and finally 72°C extension for 7 minutes.

[0042] 4. Electrophoresis detection

[0043] 4.1 Prepare gel: According to the type of molecular marker and the size of the amplified product, select agarose gel or polyacrylamide gel of appropriate concentration. For example, 3%-5% agarose gel is commonly used to detect SSR amplified products, and 6% polyacrylamide gel is commonly used to detect AFLP products. Add an appropriate amount of electrophoresis buffer, heat to dissolve, and pour into the electrophoresis tank. Insert the comb and wait for the gel to cool and solidify to form a sample loading well.

[0044] 4.2 Loading and electrophoresis: After mixing the PCR amplification product with the loading buffer, add it to the loading hole of the gel, turn on the power for electrophoresis, set the appropriate voltage and electrophoresis time to separate the amplification products by size in the gel. For example, agarose gel electrophoresis generally uses a voltage of 100-150V and electrophoresis for 30-60 minutes.

[0045] 5. Results Analysis

[0046] 5.1 Staining and imaging: After electrophoresis, the gel is stained. Common staining methods include ethidium bromide (EB) staining and silver staining, so as to facilitate observation and photographing of electrophoresis bands. For example, agarose gel can be stained with EB and then observed and photographed under ultraviolet light, while polyacrylamide gel is mostly stained with silver. After fixation, staining, and color development, a clear band image can be obtained.

[0047] 5.2 Data analysis: Analyze the molecular marker polymorphism between different samples based on the presence, size, quantity and other characteristics of the electrophoresis bands, such as calculating the genetic similarity coefficient and constructing a cluster diagram.

[0048] S4. Hybridize the parents to obtain F1 hybrids: plant the parents in a greenhouse, remove defective plants in stages during the planting process, plant the donor parent A and the recipient parent B twice, and adjust the temperature so that their flowering periods coincide. Artificially hybridize the selected parents. When hybridizing the parents, collect pollen from the male parent and fertilize it on the female ear of the female parent, and put it in a bag for isolation to ensure the hybridization effect. Harvest F1 seeds and plant them. Perform routine management on the F1 seeds during their growth period, such as fertilizing, watering, and controlling pests and diseases.

[0049] Specifically, choose soil with high adsorption activity and good water and fertilizer retention effects for planting, promote the root system of the plant to grow deep and expand the root absorption range, improve the utilization rate of water and fertilizer, and at the same time cooperate with ammonium bicarbonate, defluorinated phosphate fertilizer, kiln ash potassium fertilizer, calcium sulfate, magnesium sulfate, sodium selenite, sodium molybdate, and sodium nitrophenol to provide the necessary trace elements for the growth of fresh corn, so that the plant's absorption of nutrients is more comprehensive and uniform, and the quality of the plant is improved. The phenotypic expression of the target traits is observed, and the phenotypic selection focuses on plant height, number of ears, firmness and disease resistance level, etc., and molecular markers are combined with phenotypes for selection. Phenotypic selection takes disease resistance and yield as important selection indicators, and further selects the best and eliminates the worst according to the results of molecular detection to obtain high-quality plants that meet the breeding goals. This step is to verify the accuracy of molecular marker selection, because marker detection may have errors or incomplete linkage.

[0050] S5. Molecular marker detection: leaf tissues are collected from F1 plants, genomic DNA is extracted, and then the selected molecular markers are used to perform genotype detection on F1 individuals to determine whether they contain the target gene and screen out heterozygous individuals containing the target gene. Specifically, a sampler is selected to collect leaf tissues from F1 plants, and the sample is ground using a grinder. Then, 50 μl of NaOH solution is added to the ground sample, and the sample is placed in a water bath at 98°C-100°C for 10 minutes. The sample is taken out of the boiling water, and 50 μl of TE solution is added to neutralize the alkalinity in step S3. This mixed solution contains sample DNA for genotype detection;

[0051] S6. Backcrossing or self-pollination: The selected F1 generation heterozygous individuals are self-pollinated or backcrossed with the recipient parent to obtain F2 generation or BC1 generation seeds. After planting, DNA is extracted to detect molecular markers. Based on the test results, individuals containing multiple target genes and with a high background recovery rate are selected;

[0052] S7. Multi-generation selection and identification: Repeat the process of backcrossing or self-pollination and molecular marker detection. After multiple generations of selection, the homozygosity of the target gene and the aggregation of excellent genes are gradually increased, and the combination of these beneficial genes in the breeding material is fixed. At the same time, combined with field phenotypic identification, stable strains with excellent agronomic traits and homozygous target genes are screened out.

[0053] In the present invention, the method for efficiently breeding fresh corn using molecular markers is further described by experimental examples:

[0054] 1. Parent selection

[0055] Fresh corn varieties A and B were selected as parent materials. A is a high-yield variety but slightly weaker disease resistance, and B is a variety with strong disease resistance but relatively low yield. It is hoped that the advantages of the two can be combined through hybrid breeding and the use of molecular markers to assist selection.

[0056] 2. Planting process

[0057] The parents are planted in a greenhouse, the donor parent A and the recipient parent B are planted twice, and the temperature is adjusted to make their flowering periods coincide. The selected parents are artificially hybridized, and F1 generation seeds are harvested and planted. The genotype of the F1 generation individuals is detected using the selected molecular markers to determine whether they contain the target gene, and the heterozygous individuals containing the target gene are screened out. The screened F1 generation heterozygous individuals are self-pollinated or backcrossed with the recipient parent to obtain F2 generation or BC1 generation seeds. After planting, DNA is further extracted to detect molecular markers, and based on the test results, the seeds containing multiple The process of backcrossing or self-pollination and molecular marker detection is repeated. After multiple generations of selection, the homozygosity of the target gene and the aggregation degree of excellent genes are gradually increased, and the combination of these beneficial genes in the breeding materials is fixed. At the same time, combined with field phenotypic identification, phenotypic selection focuses on plant height, number of ears, fruitfulness and disease resistance level, and molecular markers are combined with phenotypes for selection. Phenotypic selection takes disease resistance and yield as important selection indicators, and further selects the best and eliminates the worst according to the results of molecular detection, and screens out stable strains with excellent agronomic traits and homozygous target genes.

[0058] The planting conditions for each generation are roughly the same. Choose land with fertile soil, deep soil layer, good air permeability and good drainage, and the pH value of 6-7 is appropriate. Deeply plow the soil about 20-30 cm, rake the soil finely, rake flat, and remove debris. If the soil fertility is insufficient, base fertilizer can be applied in combination with land preparation. Generally, 1000-2000 kg of organic fertilizer, 30-40 kg of superphosphate, and 10-15 kg of potassium sulfate are applied per mu. When the temperature of the 5-10 cm soil layer on the surface is stable above 10°C, sow with equal row spacing or wide and narrow rows. The equal row spacing is generally about 60 cm, and the wide row of wide and narrow rows is 80-90 cm, and the narrow row is 30-40 cm. Sow 2-3 seeds in each hole, sow to a depth of 3-5 cm, and cover the soil and compact it after sowing. When fresh corn grows to 3-4 leaves, thin out the seedlings and remove weak and diseased seedlings; when the leaves are 5-6, the seedlings are fixed, and one strong seedling is left in each hole. During the growth period, 2-3 inter-cultivation is carried out to a depth of 5-10 cm. At the same time, weeds are removed. Manual weeding or herbicides such as acetochlor can be used. Fresh corn requires a large amount of fertilizer. In addition to base fertilizer, topdressing is also required. Apply 10-15 kg of urea per mu during the jointing stage, 20-25 kg of urea and 10-15 kg of potassium chloride during the large trumpet stage. Fresh corn needs sufficient water to grow, especially during the tasseling and filling stages. In case of drought, water should be applied in time. The amount of water each time should be enough to wet the soil 20-30 cm. The fresh corn plant's bracts turn yellow and loose, the kernels become hard and shiny, and the milk line disappears before harvesting. After harvesting, it should be dried or dried to reduce the moisture content of the kernels to about 14% for storage.

[0059] 3. Test results

[0060] Molecular marker detection:

[0061]

[0062] Test data:

[0063]

[0064]

[0065]

[0066] Field phenotyping:

[0067]

[0068]

[0069] It can be seen from the examples that the use of molecular markers to assist in the breeding of fresh-eating corn has the following beneficial effects:

[0070] 1. The present invention improves selection accuracy: Traditional breeding based on phenotypic selection is easily interfered by environmental factors, while molecular markers directly detect DNA, which is not affected by the environment and developmental stage, and can accurately identify the existence and transmission of target genes. Traditional fresh corn breeding has a long cultivation cycle, and molecular marker-assisted selection can screen the target trait genotype in the seedling stage or early generations without waiting until the plant matures and expresses the phenotype, shortening the breeding cycle and launching excellent varieties more quickly.

[0071] 2. The present invention enhances breeding predictability: With the help of molecular markers, the genotype combination and target trait performance of hybrid offspring can be understood in advance, breeding programs can be rationally planned, and the probability of obtaining excellent varieties can be increased, such as predicting hybrid vigor and selecting advantageous combinations for hybrid breeding.

[0072] 3. The present invention can select multiple target traits at the same time, and can also quickly screen out individuals with target genotypes in a large population, reducing the workload of field trials and phenotypic identification, allowing breeders to concentrate resources on cultivating superior varieties.

[0073] 4 The present invention can accurately identify the purity and authenticity of fresh corn varieties through molecular markers, prevent counterfeit and inferior seeds from entering the market, and ensure seed quality and agricultural production safety, such as detecting the proportion of genetic components of parents in hybrids to ensure that the varieties meet quality standards.

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for efficient breeding of fresh corn using molecular markers, characterized in that: The following steps are involved: S1, target trait confirmation: determine the target trait that needs to be improved, and locate the gene or quantitative trait locus QTL associated with the target trait through linkage analysis or genome-wide association study (GWAS); S2, development of molecular markers: development of tightly linked molecular markers for located genes or QTLs; S3. Screening of breeding materials: Use the developed molecular markers to screen breeding materials, identify donor parents containing target genes and recipient parents with excellent comprehensive traits, and molecularly mark the target genes in the parents; S4, hybridizing the parents to obtain F1 hybrids: artificially hybridizing the selected parents, harvesting F1 seeds, planting them, and observing the phenotypic performance of the target traits; S5. Molecular marker detection: Collect leaf tissue from F1 generation plants, extract genomic DNA, and then use selected molecular markers to perform genotype detection on F1 generation individuals to determine whether they contain the target gene and screen out heterozygous individuals containing the target gene; S6. Backcrossing or self-pollination: The selected F1 generation heterozygous individuals are self-pollinated or backcrossed with the recipient parent to obtain F2 generation or BC1 generation seeds. After planting, DNA is extracted to detect molecular markers. Based on the test results, individuals containing multiple target genes and with a high background recovery rate are selected; S7. Multi-generation selection and identification: Repeat the process of backcrossing or self-pollination and molecular marker detection. After multiple generations of selection, the homozygosity of the target gene and the aggregation of excellent genes are gradually increased, and the combination of these beneficial genes in the breeding material is fixed. At the same time, combined with field phenotypic identification, stable strains with excellent agronomic traits and homozygous target genes are screened out.

2. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In the S5, a sampler is selected to collect leaf tissue from the F1 generation plants, and the sample is ground using a grinder. Then, 50 μl of NaOH solution is added to the ground sample, and the sample is bathed in 98° C.-100° C. water for 10 minutes. The sample is taken out of the boiling water, and 50 μl of TE solution is added to neutralize the alkalinity in step S3. This mixed solution contains sample DNA for genotype detection.

3. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S3, when selecting parents, factors such as the relationship between the parents and the combining ability are comprehensively considered to obtain good hybrid advantages.

4. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S2, the type of molecular marker is SSR marker.

5. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S4, the parents are planted in a greenhouse, the donor parent A and the recipient parent B are planted twice, and the temperature is adjusted to make their flowering periods coincide. When the parents are crossed, the pollen of the male parent is collected and fertilized on the female ear of the female parent, and then bagged for isolation.

6. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S4, phenotypic selection focuses on plant height, number of ears, fruitfulness and disease resistance level, and combines molecular markers with phenotypes for selection. Phenotypic selection uses disease resistance and yield as important selection indicators, and further selects and eliminates the inferior based on the results of molecular testing to obtain high-quality plants that meet the breeding goals.

7. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S1, there may be multiple target genes.

8. The method for efficient breeding of fresh-eating corn using molecular markers as aid of claim 1, characterized in that: In S4, conventional management is performed on the F1 generation seeds during their growth period, such as fertilization, watering, and pest and disease control.

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