CN119391902A - A method for efficient breeding of fresh-eating corn using molecular markers - Google Patents
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Abstract
本发明公开了一种利用分子标记辅助鲜食玉米高效育种方法，包括S1，目标性状确认：确定需要改良的目标性状，并通过连锁分析或全基因组关联研究(GWAS)定位与目标性状相关的基因或数量性状位点(QTL)。本发明设计合理，能准确鉴别目标基因的存在与传递，提高选择的准确性，且分子标记辅助选择可在幼苗期或早期世代对目标性状基因型筛选，缩短育种周期，更快推出优良品种。借助分子标记可提前了解杂交后代的基因型组合及目标性状表现，合理规划育种方案，提高获得优良品种的概率，并且能同时对多个目标性状进行选择，还可在大量群体中快速筛选出具有目标基因型的个体，减少田间试验和表型鉴定工作量，让育种者集中资源培育优良品种。
The present invention discloses a method for efficient breeding of fresh corn using molecular markers, including S1, target trait confirmation: determining the target trait that needs to be improved, and locating the gene or quantitative trait locus (QTL) related to the target trait through linkage analysis or genome-wide association study (GWAS). The present invention is reasonably designed, can accurately identify the existence and transmission of the target gene, 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 in a large population, reducing the workload of field trials and phenotypic identification, allowing breeders to concentrate resources on cultivating superior varieties.
Description
Technical Field
The invention relates to the technical field of molecular marker assisted breeding, in particular to a high-efficiency breeding method for fresh corn by using a molecular marker.
Background
Molecular marker assisted breeding is a modern breeding technology, and the basic principle is that an individual with a target character is indirectly selected by detecting a molecular marker by utilizing the close linkage relation between the molecular marker and the target character gene. The molecular markers may be variations in DNA sequence, such as Single Nucleotide Polymorphisms (SNPs) or Simple Sequence Repeats (SSRs), which are numerous in number and mostly co-dominant in the genome, independent of environmental conditions.
The molecular marker assisted breeding has the advantages of being rapid, accurate and free from interference of environmental conditions, can remarkably improve the breeding efficiency, shortens the breeding period and overcomes the difficulty in the traditional breeding method. Unlike transgenic breeding, molecular marker assisted breeding does not involve direct modification of genes, but rather, improves varieties by marker assisted selection, and thus does not involve the safety problem of transgenes.
The fresh corn is annual herb of the genus corn of the family Gramineae. The fresh corn is a cross-pollinated plant of a annual hermaphrodite plant, has high plant size and strong stem, is an important grain crop and feed crop, is also a crop with the highest total yield worldwide, and has the planting area and the total yield inferior to those of rice and wheat. The fresh corn is praised as a longevity food, contains rich proteins, fats, vitamins, microelements, cellulose and the like, and has great potential for developing high-nutrition and high-biological functional food. However, due to the fact that the hereditary is complex, the variation variety is rich, the defects of overlong period, overlarge variation coefficient and influence on the growth and development of offspring exist in the conventional breeding process, and therefore the efficient breeding method for the fresh corn with the assistance of the molecular marker is provided for solving the problems.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a high-efficiency breeding method for fresh corn by using molecular markers, which can accurately identify the existence and transmission of target genes, improve the accuracy of selection, and can screen target trait genotypes in seedling stage or early generation by molecular marker-assisted selection, shorten breeding period and promote good varieties more quickly. The genotype combination and target character expression of the filial generation can be known in advance by means of molecular markers, 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, the workload of field experiments and phenotype identification is reduced, and breeders can concentrate resources to cultivate the good varieties.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for efficiently breeding fresh corn by using molecular markers comprises the following steps:
s1, confirming a target trait, namely determining the target trait to be improved, and positioning a gene or Quantitative Trait Locus (QTL) related to the target trait through linkage analysis or whole genome association study (GWAS);
S2, developing a molecular marker, namely developing a closely linked molecular marker aiming at a localized gene or QTL;
s3, screening the breeding materials by using developed molecular markers, identifying donor parents containing target genes and acceptor parents with excellent comprehensive properties, and carrying out molecular markers on the target genes in the parents;
S4, hybridizing the parents to obtain F1 generation hybrid, namely manually hybridizing the selected parents, harvesting F1 generation seeds, planting, and observing the phenotype expression of the target character;
S5, molecular marker detection, namely collecting leaf tissues from F1 generation plants, extracting genome DNA, carrying out genotype detection on F1 generation individuals by using a selected molecular marker, determining whether the F1 generation individuals contain target genes, and screening heterozygous individuals containing the target genes;
S6, backcrossing or selfing, namely selfing or backcrossing the screened F1 generation heterozygous individuals with a receptor parent to obtain F2 generation or BC1 generation seeds, continuously extracting DNA detection molecular markers after planting, and selecting individuals with a plurality of target genes and high background recovery rate according to detection results;
S7, selecting and identifying for multiple generations, namely repeating backcross or selfing and molecular marker detection processes, gradually increasing the homozygosity of the target genes and the polymerization degree of the excellent genes through multiple generations of selection, fixing the combination of the beneficial genes in breeding materials, and screening stable strains with excellent agronomic characters and homozygosity of the target genes by combining field phenotype identification.
Preferably, in the step S5, a sampler is selected to collect leaf tissue from F1 generation plants, a grinder is used for grinding the sample, 50 μl of NaOH solution is added into the ground sample, the sample is taken out from boiling water after 10min in water at 98-100 ℃,50 μl of TE solution is added for neutralizing the alkalinity in the step S3, and the mixed solution contains sample DNA for genotype detection.
Preferably, in the step S3, when the parents are selected, factors such as relationships between the parents and the mating force are comprehensively considered so as to obtain good heterosis.
Preferably, in S2, the type of the molecular marker is an SSR marker.
Preferably, in the step S4, the parents are planted in a greenhouse, the donor parent A and the acceptor parent B are planted in front and back twice, the flowering phases of the parents are met by adjusting the temperature, and when the parents are hybridized, the pollen of the male parent is collected and fed onto female ears of the female parent, and the female parent and the female ears are bagged for isolation.
Preferably, in the step S4, the phenotype selection is focused on plant height, spike number, fruiting performance and disease resistance grade, and the molecular marker is combined with the phenotype to select, the phenotype selection uses disease resistance performance and yield as important selection indexes, and the quality is further improved according to the molecular detection result, so that a high-quality plant meeting the breeding target is obtained.
Preferably, in S1, the target gene may be plural.
Preferably, in S4, the F1 generation seed is conventionally managed during its growth, such as fertilization, watering, and pest control.
Compared with the prior art, the invention has the beneficial effects that:
1. The traditional breeding is easy to be interfered by factors such as environment and the like according to phenotype selection, the molecular marker directly detects DNA, is not influenced by the environment and development stage, can accurately identify the existence and transmission of a target gene, improves the accuracy of selection, and can screen the genotype of the target trait in a seedling stage or early generation without waiting until the plant is mature, shortens the breeding period and pushes out good varieties more quickly.
2. The genotype combination and target character expression of the filial generation can be known in advance by means of molecular markers, a breeding scheme is reasonably planned, the probability of obtaining good varieties is improved, such as predicting heterosis, selecting dominant combinations for cross breeding, simultaneously selecting a plurality of target characters, rapidly screening individuals with target genotypes in a large number of groups, reducing the workload of field experiments and phenotype identification, and enabling breeders to concentrate resources to cultivate the good varieties.
Drawings
Fig. 1 is a schematic flow chart of a method for efficiently breeding fresh corn by using molecular markers.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Referring to fig. 1, a method for efficiently breeding fresh corn with the assistance of molecular markers comprises the following steps:
s1, confirming target traits, namely determining the target traits to be improved, and positioning genes or Quantitative Trait Loci (QTL) related to the target traits through linkage analysis or whole genome association study (GWAS), wherein a plurality of target genes can be selected, and the target genes are selected to be high in pest resistance and high in yield.
S2, developing a molecular marker, namely developing a closely linked molecular marker aiming at a localized gene or QTL.
S3, screening breeding materials: screening breeding materials by using developed molecular markers, identifying donor parents containing target genes and acceptor parents with good comprehensive properties, carrying out molecular markers on the target genes in the parents, comprehensively considering factors such as relatedness and combining force among the parents when selecting the parents to obtain good hybrid vigor, selecting a fresh corn variety with high disease and pest resistance and general yield as a donor parent A, selecting a variety with high yield and weak disease and pest resistance as an acceptor parent B, selecting a fresh corn variety with high disease and pest resistance and general yield as the donor parent A, and selecting a variety with high yield and weak disease and pest resistance as an acceptor parent B, wherein the target genes can also be other properties such as lodging resistance, drought resistance, plant type, and flourishing property;
in the application, the molecular marker is SSR marker, and the specific steps are as follows:
a parent biological sample containing the target DNA is collected and prepared for the required experimental reagents and instrumentation.
DNA extraction and quality detection
1.1DNA extraction, namely extracting DNA in a sample by using a proper method, such as a CTAB method, an SDS method or a DNA extraction kit, and the like, so as to obtain pure and complete DNA for subsequent experiments.
1.2 Quality detection, namely measuring absorbance of DNA at wavelengths of 260nm and 280nm by an ultraviolet spectrophotometer, calculating an OD260/OD280 ratio, evaluating DNA purity, and simultaneously detecting concentration and integrity of the DNA to ensure that the DNA meets the requirements of subsequent experiments.
2. Primer design and Synthesis
2.1 Designing a primer, namely designing a specific primer aiming at a selected molecular marker type and a target gene or sequence, and designing by means of professional software so as to ensure that parameters such as the specificity, annealing temperature and the like of the primer are proper.
2.2 Synthesis of primers the designed primer sequences were submitted to professional biological companies for synthesis.
PCR amplification
3.1 Preparing a reaction system, namely sequentially adding a proper amount of DNA template, primer, dNTPs, taq enzyme, PCR buffer solution and the like into a PCR tube, adjusting the dosage of each component according to different primer and template concentrations, and finally adding sterile water to complement the reaction system to a required volume.
3.2 Setting an amplification procedure by placing the PCR tube in a PCR instrument, setting a suitable amplification procedure generally comprising the steps of pre-denaturation, annealing, extension and the like, wherein the amplification procedures of different molecular markers and primers are different, for example, the amplification procedure of SSR markers is generally 94 ℃ pre-denaturation for 5min,94 ℃ denaturation for 30s,55 ℃ annealing for 30s,72 ℃ extension for 30s, 30 cycles altogether, and finally 72 ℃ extension for 7min.
4. Electrophoresis detection
4.1 Preparing gel, namely selecting agarose gel or polyacrylamide gel with proper concentration according to the type of molecular markers and the size of amplified products, for example, detecting the SSR amplified products, wherein the agarose gel is usually 3% -5%, the polyacrylamide gel is usually 6%, adding a proper amount of electrophoresis buffer solution into the AFLP products, heating and dissolving the AFLP products, pouring the AFLP products into an electrophoresis tank, inserting a comb, and forming a sample adding hole after the gel is cooled and solidified.
4.2 Sample loading and electrophoresis, namely mixing the PCR amplification product with a sample loading buffer solution, adding the mixture into a sample loading hole of the gel, switching on a power supply for electrophoresis, and setting proper voltage and electrophoresis time to separate the amplification product in the gel according to the size, wherein the voltage of 100-150V is generally adopted for agarose gel electrophoresis, and the electrophoresis is carried out for 30-60min.
5. Analysis of results
5.1 Dyeing and imaging, namely after electrophoresis, dyeing the gel, wherein common dyeing methods include Ethidium Bromide (EB) dyeing, silver dyeing and the like, so as to facilitate observation and photographing and recording of electrophoresis strips, for example, the agarose gel can be used for observing and photographing under an ultraviolet lamp after the EB dyeing, and the polyacrylamide gel is mostly silver-dyed, and clear strip images are obtained after the steps of fixing, dyeing, color development and the like.
And 5.2, analyzing the molecular marker polymorphism between different samples according to the characteristics of the existence, the size, the number and the like of the electrophoresis bands, such as calculating a genetic similarity coefficient, constructing a cluster map and the like.
S4, parent hybridization to obtain F1 generation hybrid, namely planting the parent in a greenhouse, removing defective plant individuals in stages in the planting process, planting the donor parent A and the acceptor parent B for two times before and after each other, adjusting the temperature to meet the flowering period, manually hybridizing the selected parent, collecting pollen of the parent, putting the pollen of the parent on female ears, bagging and isolating to ensure the hybridization effect, harvesting F1 generation seeds, planting, and carrying out conventional management such as fertilization, watering and pest control during the growth of the F1 generation seeds.
Specifically, planting is carried out in soil with high adsorption activity and good water and fertilizer retention effect, the plant root system is promoted to be pricked down, the root system absorption range is enlarged, the water and fertilizer utilization rate is improved, and meanwhile, the soil is matched with ammonium bicarbonate, defluorinated phosphate fertilizer, kiln ash potassium fertilizer, calcium sulfate, magnesium sulfate, sodium selenite, sodium molybdate and compound sodium nitrophenolate, so that required microelements are provided for the growth of fresh corn, the plant absorbs nutrients more comprehensively and uniformly, the plant quality is improved, the phenotype performance of the target property is observed, the phenotype selection is focused on plant height, spike number, fruiting performance, disease resistance and disease resistance grade and the like, the molecular marker is combined with the phenotype to select, the disease resistance performance and yield are used as important selection indexes, and the quality plant meeting the breeding target is further selected and washed and degraded according to the molecular detection result.
S5, molecular marker detection, namely collecting leaf tissues from F1 generation plants, extracting genome DNA, carrying out genotype detection on F1 generation individuals by using a selected molecular marker, determining whether the F1 generation individuals contain target genes, screening heterozygous individuals containing the target genes, specifically, collecting the leaf tissues from the F1 generation plants by using a sampler, grinding a sample by using a grinder, adding 50 mu l of NaOH solution into the ground sample, carrying out water bath for 10min at 98-100 ℃, taking the sample out of boiling water, adding 50 mu l of TE solution, neutralizing the alkalinity in the step S3, wherein the mixed solution contains sample DNA and is used for genotype detection;
S6, backcrossing or selfing, namely selfing or backcrossing the screened F1 generation heterozygous individuals with a receptor parent to obtain F2 generation or BC1 generation seeds, continuously extracting DNA detection molecular markers after planting, and selecting individuals with a plurality of target genes and high background recovery rate according to detection results;
S7, selecting and identifying for multiple generations, namely repeating backcross or selfing and molecular marker detection processes, gradually increasing the homozygosity of the target genes and the polymerization degree of the excellent genes through multiple generations of selection, fixing the combination of the beneficial genes in breeding materials, and screening stable strains with excellent agronomic characters and homozygosity of the target genes by combining field phenotype identification.
In the invention, the efficient breeding method of the fresh corn assisted by the molecular marker is further described by experimental examples:
1. Parental selection
Fresh corn varieties A, B are selected as parent materials, A is a high-yield and slightly weak-disease-resistance variety, B is a high-disease-resistance and relatively low-yield variety, the advantages of the two varieties are expected to be combined through hybridization breeding, and molecular markers are utilized for auxiliary selection.
2. Planting process
Planting parents in a greenhouse, planting donor parents A and acceptor parents B twice before and after the parents are planted, regulating the temperature to meet the flowering phase of the parents, artificially hybridizing the selected parents, harvesting F1 generation seeds, planting, detecting genotypes of F1 generation individuals by using selected molecular markers, determining whether the F1 generation individuals contain target genes, screening heterozygous individuals containing the target genes, carrying out selfing or backcrossing with the acceptor parents on the screened F1 generation heterozygous individuals to obtain F2 generation or BC1 generation seeds, continuously extracting DNA detection molecular markers after planting, selecting individuals simultaneously containing a plurality of target genes and having high background recovery rate according to detection results, repeating the backcross or selfing and molecular marker detection processes, gradually increasing the homozygosity of the target genes and the polymerization degree of good genes through multiple generation selection, fixing the combination of the good genes in breeding materials, simultaneously combining field phenotype identification, carrying out phenotype selection on the high-forming spike number, the fruit bearing performance, the grade and the like, combining the molecular markers with the phenotype selection, carrying out selection, taking the phenotype selection and the good gene performance as important selection results, and further selecting good and stable characteristics according to the good and good quality characteristics of the detection results.
The planting conditions are approximately the same in each generation, and the soil with rich soil, deep soil layer, good air permeability and good drainage is selected, and the pH value is preferably 6-7. Deep ploughing soil for about 20-30 cm, raking soil blocks finely and flatly, and removing sundries. If the soil fertility is insufficient, the soil preparation can be combined with the base fertilizer application, and 1000-2000 kg of organic fertilizer, 30-40 kg of calcium superphosphate, 10-15 kg of potassium sulfate and the like are generally applied per mu. Sowing when the soil layer temperature of the earth surface is 5-10 cm and is stabilized at 10 ℃ or above, sowing by adopting equal row spacing or wide and narrow row spacing, wherein the equal row spacing is about 60 cm, the wide row spacing of the wide and narrow row is 80-90 cm, and the narrow row is 30-40 cm. 2-3 seeds are sowed in each hole, the sowing depth is 3-5 cm, and soil is covered and compacted after sowing. Thinning when the fresh corn grows to 3-4 leaves, removing weak seedlings and diseased seedlings, and thinning when the corn grows to 5-6 leaves, and keeping 1 strong seedling in each hole. 2-3 times of cultivation are carried out during the growth period, the depth is 5-10 cm, and meanwhile, weeds are removed, so that the weeding composition can be used for manually weeding or weeding with herbicide such as acetochlor and the like. The fresh corn needs a large amount of fertilizer, and needs additional fertilizer besides the base fertilizer. 10-15 kg of urea is applied to each mu in the jointing period, 20-25 kg of urea is applied to each mu in the large bell mouth period, and 10-15 kg of potassium chloride is applied to each mu in the large bell mouth period. Fresh corn requires sufficient moisture for growth, especially during the emasculation and grouting phases. If drought occurs, the water is watered in time, and the water amount is preferably 20-30 cm for wetting through soil each time. The bud She Bianhuang of the fresh corn plant is loose, the seeds are hard and glossy, and the corn can be harvested when the milk line disappears. And after harvesting, airing or drying to reduce the water content of the seeds to about 14 percent for storage.
3. Test results
Molecular marker detection conditions:
Test data:
Phenotypic analysis in the field:
The combination embodiment shows that the molecular marker is utilized to assist the fresh corn breeding to have the following beneficial effects:
1. The invention improves the selection accuracy, the traditional breeding is easy to be interfered by factors such as environment and the like according to phenotype selection, the molecular marker directly detects DNA, is not influenced by the environment and development stage, can accurately identify the existence and transmission of target genes, has long breeding period of traditional fresh corn, can screen target character genotypes in seedling stage or early generation by molecular marker auxiliary selection, does not need to wait until plants are mature to show phenotype, shortens breeding period, and pushes out good varieties more quickly.
2. The invention enhances the predictability of breeding, namely, the genotype combination and target character expression of the filial generation can be known in advance by means of molecular markers, the breeding scheme is reasonably planned, the probability of obtaining good varieties is improved, such as predicting heterosis, and the dominant combination is selected for hybridization breeding.
3. The invention can select a plurality of target characters at the same time, and can also rapidly screen individuals with target genotypes in a large number of groups, thereby reducing the workload of field test and phenotype identification and enabling breeders to concentrate resources to cultivate good varieties.
The invention can accurately identify the purity and the authenticity of the fresh corn variety by molecular marking, prevent counterfeit seeds from entering the market, ensure the seed quality and the agricultural production safety, such as detecting the genetic component proportion of parents in the hybrid seeds, and ensure the variety to meet the quality standard.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (8)
1. The efficient breeding method for the fresh corn assisted by the molecular marker is characterized by comprising the following steps of:
S1, confirming target traits, namely determining the target traits to be improved, and positioning genes or Quantitative Trait Loci (QTL) related to the target traits through linkage analysis or whole genome association research (GWAS);
S2, developing a molecular marker, namely developing a closely linked molecular marker aiming at a localized gene or QTL;
s3, screening the breeding materials by using developed molecular markers, identifying donor parents containing target genes and acceptor parents with excellent comprehensive properties, and carrying out molecular markers on the target genes in the parents;
S4, hybridizing the parents to obtain F1 generation hybrid, namely manually hybridizing the selected parents, harvesting F1 generation seeds, planting, and observing the phenotype expression of the target character;
S5, molecular marker detection, namely collecting leaf tissues from F1 generation plants, extracting genome DNA, carrying out genotype detection on F1 generation individuals by using a selected molecular marker, determining whether the F1 generation individuals contain target genes, and screening heterozygous individuals containing the target genes;
S6, backcrossing or selfing, namely selfing or backcrossing the screened F1 generation heterozygous individuals with a receptor parent to obtain F2 generation or BC1 generation seeds, continuously extracting DNA detection molecular markers after planting, and selecting individuals with a plurality of target genes and high background recovery rate according to detection results;
S7, selecting and identifying for multiple generations, namely repeating backcross or selfing and molecular marker detection processes, gradually increasing the homozygosity of the target genes and the polymerization degree of the excellent genes through multiple generations of selection, fixing the combination of the beneficial genes in breeding materials, and screening stable strains with excellent agronomic characters and homozygosity of the target genes by combining field phenotype identification.
2. The method for efficient breeding of corn with molecular marker as claimed in claim 1, wherein in S5, a sampler is selected to collect leaf tissue from the F1 plant, the sample is ground by a grinder, 50 μl NaOH solution is added to the ground sample, the sample is taken out from boiling water in water at 98-100 ℃ for 10min, 50 μl TE solution is added to neutralize the alkalinity in S3, the mixed solution contains sample DNA for genotype detection.
3. The method for efficient breeding of corn with molecular marker as claimed in claim 1, wherein in S3, the parents are selected while comprehensively considering the factors such as the relationship between the parents and the mating force, so as to obtain good heterosis.
4. The method for high-efficiency breeding of fresh corn by using molecular markers according to claim 1, wherein in the step S2, the type of the molecular markers is SSR markers.
5. The method for efficient breeding of corn with molecular marker assisted fresh eating according to claim 1, wherein in S4, the parent is planted in a greenhouse, the donor parent a and the acceptor parent B are planted twice before and after each other, the flowering phases are met by adjusting the temperature, and when the parent is hybridized, the pollen of the male parent is collected and fed to the female parent, and the female parent are isolated by bagging.
6. The method for efficient breeding of corn with molecular marker as claimed in claim 1, wherein in S4, the phenotype selection is focused on plant height, spike number, fruiting performance and disease resistance level, the molecular marker is combined with the phenotype for selection, the phenotype selection uses disease resistance performance and yield as important selection indexes, and the quality of plants meeting the breeding objective is further selected according to the molecular detection result.
7. The method for efficient breeding of corn with molecular marker as claimed in claim 1, wherein in S1, the target genes may be plural.
8. The method for efficient breeding of fresh corn using molecular markers according to claim 1, wherein in S4, the F1 generation seeds are routinely managed during growth, such as fertilization, watering, and pest control.
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