High-efficiency breeding method for corn single chromosome substitution line and application of high-efficiency breeding method

Abstract

The invention discloses an efficient breeding method for a corn single chromosome substitution line and application of the efficient breeding method. The invention provides a single Chromosome Substation Line (SCSL) breeding method, which utilizes SNP (Single Nucleotide Polymorphism) and HTP (Hypertext Transfer Protocol) type molecular markers, adopts a'simplified marker-gene chip 'grading evaluation strategy, and is rapid, efficient, accurate and high in throughput. Taking a male parent inbred line Jing92 and a female parent inbred line Jing724 of a corn main push variety Jingke 968 as parent materials, a first corn SCSL population is successfully created. Through the implementation of the method, the problems that traditional genetic population creation is low in efficiency, large in resource consumption, complex in genetic background and incompatible in genetic positioning and breeding are solved, and the novel population creation method which is efficient, economical, single in background and compatible in genetic positioning and breeding is provided. And a brand-new scheme support is provided for genetic research and precise breeding of corn.

Description

Technical Field

[0001] The present invention belongs to the field of plant breeding, and particularly relates to a method for efficiently selecting and breeding maize single chromosome substitution lines and its application. Background Art

[0002] Maize is the most widely planted crop for both food and feed worldwide and is also a typical model crop in genetic breeding research. The creation of maize genetic populations provides an important research basis for the excavation of excellent genes and the genetic improvement of excellent traits. In the creation of genetic populations, maize inbred lines with obvious trait differences are usually preferentially selected as parents. After years and multiple generations of hybridization, backcross (BC), and self-crossing processes, different types of genetic populations are finally produced. Among them, Chromosomal Segment Substitution Lines (CSSL) as a typical permanent genetic population are widely used in maize genetic basic research.

[0003] Currently, simple sequence repeats (SSR) are mostly used in the creation of maize CSSL, and the throughput of marker numbers and sample detection is low, which is likely to cause the loss of target individual plants. At the same time, as a genetic population, in the process of creating CSSL, each individual only retains part of the exogenous chromosome segment, and it is impossible to achieve full-genome coverage of the exogenous segment. Therefore, it is necessary to retain as many individuals that meet the requirements as possible to ensure the full-genome coverage of the exogenous chromosome segment. Most CSSL can only be used for gene mapping of specific differential traits, cannot take into account the simultaneous mapping of multiple traits, and cannot be used for maize genetic breeding. Therefore, it is urgent to create a genetic population that combines mapping and breeding, and develop a set of rapid, efficient, accurate, and high-throughput methods for selecting and breeding chromosome substitution lines as a new tool for maize genetic basic research and variety improvement to ensure the development and innovation of maize breeding. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for efficiently selecting and breeding maize single chromosome substitution lines and its application.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a set of SNP molecular marker sets for efficiently selecting and breeding maize single chromosome substitution lines, and the SNP molecular markers are selected from any one or more of the following in the set:

[0007] Chr 1:11486898 (B73_v3) A / G, Chr 1:35047742 (B73_v3) A / G, Chr 1:156554418 (B73_v3) G / T, Chr 1:290379031 (B73_v3) A / G, Chr 2:1283337 (B73_v3) C / T, Chr 2:109563977 (B73_v3) G / T, Chr 2:223977329 (B73_v3) G / T, Chr 3:15222626 (B73_v3) G / T, Chr 3:88400490 (B73_v3) A / G, Chr 3:212050056 (B73_v3) A / C, Chr 4:18832692 (B73_v3) A / C, Chr 4:128874467 (B73_v3) G / T, Chr 4:240891846 (B73_v3) A / C, Chr 5:6881508 (B73_v3) C / T, Chr 5:88226331 (B73_v3) C / T, Chr 5:204879477 (B73_v3) A / G, Chr 6:4843862 (B73_v3) T / C, Chr 6:39822980 (B73_v3) T / C, Chr 6:158855834 (B73_v3) A / G, Chr 7:13214477 (B73_v3) A / C, Chr 7:88150665 (B73_v3) G / T, Chr 7:175252106 (B73_v3) A / G, Chr 8:7624060 (B73_v3) G / T, Chr 8:59490913 (B73_v3) A / G, Chr 8:165927215 (B73_v3) T / G, Chr 9:11505101 (B73_v3) C / T, Chr 9:61772044 (B73_v3) C / T, Chr 9:155054805 (B73_v3) A / G, Chr 10:2802839 (B73_v3) A / C, Chr 10:39960290 (B73_v3) C / T, Chr 10:130399836 (B73_v3) A / G.

[0008] The second aspect of the present invention provides primers for detecting the SNP molecular markers described in the first aspect of the present invention, and the sequences of the primers are shown as any one or more of SEQ ID NO: 1-SEQ ID NO: 93.

[0009] The third aspect of the present invention provides a detection product comprising the primers described in the second aspect of the present invention.

[0010] Furthermore, the detection product includes a detection reagent or a kit.

[0011] The fourth aspect of the present invention provides the application of the SNP molecular marker described in the first aspect of the present invention, the primer described in the second aspect of the present invention, or the detection product described in the third aspect of the present invention in the breeding of maize single chromosome substitution lines.

[0012] The fifth aspect of the present invention provides a method for efficiently breeding maize single chromosome substitution lines, the method comprising: preliminarily screening target individual plants by using the SNP molecular marker described in the first aspect of the present invention, the primer described in the second aspect of the present invention, or the detection product described in the third aspect of the present invention.

[0013] Furthermore, the method further includes performing genotyping using a gene chip, and performing foreground and background selection on the backcross progeny obtained by preliminary screening by using HTP molecular markers to screen individuals with high foreground coverage and high background recovery rate.

[0014] Furthermore, the method uses Jing 724 as the donor parent and Jing 92 as the recipient parent.

[0015] Furthermore, the method for performing foreground and background selection on the backcross progeny includes: performing genotyping using a gene chip, comparing the genotypes of the backcross progeny with those of the recipient parent, and converting them into blockified HTP molecular markers, and calculating the foreground coverage and background recovery rate of the backcross progeny.

[0016] Furthermore, the gene chip is a Maize6H-60K SNP gene chip.

[0017] Furthermore, the method for performing foreground and background selection on the backcross progeny further includes: performing data noise reduction before and after the genotype is converted into an HTP molecular marker.

[0018] Furthermore, data noise reduction is performed based on a data noise reduction algorithm of a sliding window.

[0019] Furthermore, the foreground coverage and background recovery rate include the coverage rate of exogenous target chromosomes and the genetic background recovery rate of non-target chromosomes.

[0020] Furthermore, the screening condition for the target individual plant is the backcross progeny with a relatively high coverage rate of exogenous target chromosomes and a top 5 non-target chromosome background recovery rate.

[0021] Furthermore, the specific steps of the method include:

[0022] (1) Using maize inbred line Jing 92 as the recipient parent and Jing 724 as the donor parent, performing hybridization to obtain F1 progeny, using Jing 92 as the female parent and F1 progeny as the male parent to obtain BC1F1 backcross progeny;

[0023] (2) Compare the genotypes of BC1F1 individuals with those of the recipient parent using the polymorphic SNP loci identified by genotyping with the Maize6H-60K SNP gene chip between the two parents, and convert them into block-based HTP molecular markers. Calculate the foreground coverage rate and background recovery rate of BC1F1 individuals.

[0024] (3) Use Jing 92 as the female parent and the BC1F1 individuals screened in step (2) as the male parent for backcrossing to obtain BC2F1 backcross progeny. Screen using SNP molecular markers and the Maize6H-60K SNP gene chip, and calculate the foreground coverage rate and background recovery rate of BC2F1 individuals.

[0025] (4) Use Jing 92 as the female parent and the BC2F1 individuals screened in step (3) as the male parent for backcrossing to obtain BC3F1 backcross progeny. Screen using SNP molecular markers and the Maize6H-60K SNP gene chip, calculate the foreground coverage rate and background recovery rate of BC3F1 individuals, and self-cross the screened BC3F1 individuals to obtain BC3F2 progeny.

[0026] (5) Screen using SNP molecular markers and the Maize6H-60K SNP gene chip, calculate the foreground coverage rate and background recovery rate of BC3F2 individuals, and respectively determine the single chromosome substitution lines for each chromosome of Jing 92.

[0027] (6) Self-cross BC3F2 individuals to obtain homozygous single chromosome substitution lines.

[0028] The sixth aspect of the present invention provides the application of the single chromosome substitution lines obtained by the SNP molecular markers described in the first aspect of the present invention, the primers described in the second aspect of the present invention, the detection products described in the third aspect of the present invention, or the method described in the fifth aspect of the present invention in maize genetics research.

[0029] The seventh aspect of the present invention provides the application of the single chromosome substitution lines obtained by the SNP molecular markers described in the first aspect of the present invention, the primers described in the second aspect of the present invention, the detection products described in the third aspect of the present invention, or the method described in the fifth aspect of the present invention in maize genetic breeding.

[0030] The eighth aspect of the present invention provides the application of the single chromosome substitution lines obtained by the SNP molecular markers described in the first aspect of the present invention, the primers described in the second aspect of the present invention, the detection products described in the third aspect of the present invention, or the method described in the fifth aspect of the present invention in the creation of new maize F2, DH, and CSSL populations.

[0031] Advantages and beneficial effects of the present invention:

[0032] The present application provides a method for breeding single chromosome substitution lines (SCSLs) quickly, efficiently, accurately, and with high throughput, using SNP and HTP type molecular markers and adopting a "simplified marker - gene chip" hierarchical evaluation strategy. Using the male inbred line Jing 92 and the female inbred line Jing 724 of the main maize variety Jingke 968 as parental materials, the first SCSL population of maize was successfully created. Compared with the prior art, it has the following significant advantages:

[0033] 1. High - efficiency and accurate screening of target individuals: In this application, the BC populations of each generation adopt a strategy of combining high - density, high - throughput SNP gene chips and high - throughput SNP simplified molecular markers of samples to evaluate the genetic background recovery rate and the coverage rate of the target exogenous chromosome of BC individuals. Using the polymorphic SNP loci between the two parents of the BC population, a small number of SNP simplified molecular markers are developed. Genotyping the seed endosperm or leaves of a large number of BC progeny individuals using SNP simplified markers, and quickly screening out individuals with a relatively high coverage of the exogenous target chromosome. Genotyping the small - scale BC individuals screened above using a Maize6H - 60K SNP gene chip and converting them into HTP molecular markers to accurately evaluate the target chromosome coverage rate and the non - target chromosome background recovery rate. Finally, individuals meeting the breeding requirements are determined, improving the accuracy of target individual screening.

[0034] 2. Precise background evaluation algorithm: In this application, based on a sliding - window data - denoising algorithm, taking full advantage of the strong linkage degree of SNP loci within HTP markers, SNP molecular markers are converted into block - type HTP molecular markers, and the target chromosome coverage rate and the non - target chromosome background recovery rate of BC population individuals are accurately evaluated through HTP molecular markers.

[0035] 3. Significantly reducing the cost of population breeding: Through the evaluation and screening of SNP gene chips and simplified markers, the size of the field - planted population is greatly reduced. Only the target individuals screened by the gene chip need to be backcrossed and self - crossed, greatly reducing the field - planted area and the amount of pollination, and improving the resource utilization rate in the creation process. Adopting the "simplified marker - gene chip" hierarchical evaluation strategy, that is, using a small number of SNP simplified markers with high - throughput samples and low - cost genotyping to conduct a preliminary screening of a large - scale BC population, and only conducting accurate genotyping of a small number of BC individuals after the preliminary screening using a high - density SNP gene chip, achieving a high - cost - performance detection scheme, reducing the overall cost, and shortening the breeding cycle of target individuals at the same time.

[0036] By implementing the method of this application, the problems of low efficiency, high resource consumption, complex genetic background, and incompatibility between genetic mapping and breeding in traditional genetic population creation are solved. A new population creation method that is efficient, economical, has a simple background, and is compatible with genetic mapping and breeding is provided, providing new scheme support for maize genetic research and precision breeding. Brief Description of the Drawings

[0037] Figure 1 It is a genotype pattern diagram (Maize6H - 60K) of BC3F2 with 10 single - chromosome substitutions of Jing92.

[0038] Figure 2 It is a diagram for identifying the anthocyanin color development at the base of the anther glumes of maize single - chromosome substitution line SCSL02 and inbred parent Jing92. Detailed Embodiments

[0039] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0040] The present invention provides a set of SNP molecular markers for the efficient selection of maize single - chromosome substitution lines.

[0041] In some embodiments, SNP refers to Single Nucleotide Polymorphism, mainly referring to DNA sequence polymorphisms caused by variations of single nucleotides at the genomic level. The variations of the single nucleotides include variations caused by transitions, transversions, insertions, or deletions of single bases.

[0042] The present invention provides a primer for detecting the above - mentioned SNP molecular markers.

[0043] In some embodiments, the primer is a KASP (Kompetitive Allele Specific PCR) primer, and the KASP primer may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labels include but are not limited to fluorophores such as FAM, HEX; dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as DIG; luminescent, phosphorescent or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or optionally, can be charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

[0044] In a preferred embodiment, the label is selected from fluorophores.

[0045] In a specific embodiment, the fluorophore is selected from FAM, HEX.

[0046] The present invention provides a detection product comprising the above-mentioned primer.

[0047] The detection product includes a detection reagent or a kit.

[0048] In some embodiments, the kit further includes deionized water, a buffer, a preservative or a protein stabilizer. The kit may also contain the necessary components for detecting a detectable reagent (e.g., a substrate, an enzyme, etc.). The kit may also contain a control sample or a series of control samples, which can be assayed and compared with the test samples contained therein. Each component of the kit is usually encapsulated in a separate container, and all different containers are packed in a package and accompanied by an instruction manual containing the product composition, storage method, etc.

[0049] The components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, bottle, syringe or other container, in which one component can be placed, and preferably, appropriate aliquots can be made. When there is more than one component in the kit, the kit will usually also include a second, third or other additional containers in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the present application will usually also include a container for containing the reactants, sealed for commercial sale. Such a container can include a molded or blow-molded plastic container in which the required vials can be retained.

[0050] The solid support of the kit can be, for example, plastic, silicon wafer, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide. The biological sample can be, for example, animal and plant cell cultures, cell lines, tissues, organs, etc.

[0051] In some embodiments, during the creation of SCSL, through molecular marker technology, the process of identifying the introduction of the exogenous target chromosome provided by the donor parent in the population and screening out the target individual plants carrying the exogenous target chromosome is called foreground selection; the foreground coverage rate refers to the proportion of offspring individuals carrying the exogenous target chromosome segment. The process of identifying the recovery of the genetic background of the recipient parent in the population through molecular marker technology is called background selection; the background recovery rate refers to the degree of recovery of the genetic background of the recipient parent in the offspring individuals, that is, the proportion of the non-target chromosome genotype being the same as the recipient parent genotype.

[0052] In some embodiments, the maize SCSL of the present application is used as a genetic population for functional studies such as gene mapping. By comparing the trait differences between SCSL01 - SCSL10 and the parental inbred lines Jing 92 and Jing 724 in the field, the specific chromosome where the gene locus associated with the differential phenotypic trait is located can be quickly determined. The present application Figure 2 provides a typical differential phenotypic trait, that is, the anther glume base of SCSL02 shows deep red anthocyanin coloration, the anther glume base of Jing 92 shows colorless anthocyanin coloration, and its F1 offspring also show red glume bases, indicating that there is a dominant gene locus regulating anthocyanin accumulation at the base of the anther glume on chromosome 2. It can also be used as a "bridge parent" for genetic improvement such as the polymerization of excellent genes in maize. It can also be used as a basic material, and through techniques such as double haploid (DH), it can be used for the creation of new populations such as DH and CSSL.

[0053] The present invention will be further elaborated below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0054] Example

[0055] The process of creating the maize SCSL population in this application is as follows:

[0056] (1) Using maize inbred line Jing 92 as the recipient parent (female parent) and Jing 724 as the donor parent (male parent), cross them during the flowering period to obtain F1 offspring. Using Jing 92 as the female parent and the F1 offspring as the male parent, backcross them during the flowering period to obtain BC1F1 generation. Subsequently, use the Maize6H-60K SNP gene chip to evaluate the exogenous target chromosome coverage rate and non-target chromosome background recovery rate of BC1F1 individual plants.

[0057] (2) Use the magnetic bead method to extract the genomic DNA of the leaf tissues of Jing 92, Jing 724, and BC1F1 individual plants, and perform genotyping using the Maize6H-60K SNP gene chip. Based on the gene chip data, screen for SNP loci with polymorphic differences between Jing 92 and Jing 724. Using the polymorphic SNP loci between the two parents, compare the genotypes of BC1F1 individual plants with those of the recipient parent, and convert the genotypes of BC1F1 individual plants into digital codes. Based on the data noise reduction algorithm of the sliding window, perform data noise reduction on the genotype comparison results of the 10 chromosomes of BC1F1 individual plants and convert them into block HTP molecular markers. Based on the data noise reduction algorithm of the sliding window, perform data noise reduction on the HTP results, and calculate the exogenous target chromosome coverage rate and non-target chromosome genetic background recovery rate of BC1F1 individual plants. On the premise of the complete introduction of the exogenous target chromosome, sort the BC1F1 individual plants in descending order of the non-target chromosome background recovery rate, and finally select BC1F1 individual plants with an exogenous target chromosome coverage rate (foreground coverage rate) close to 100% and a non-target chromosome background recovery rate (background recovery rate) in the top 5 for each chromosome.

[0058] (3) Using Jing 92 as the female parent and the top 1 BC1F1 single plants of 10 different chromosomes screened in step (2) as the male parents respectively, backcrossing is carried out during the flowering period to obtain 10 BC2F1 populations of different chromosomes. The endosperm genomic DNA of BC2F1 single plants is extracted by the magnetic bead method. The "reduced representation library - genotyping array" hierarchical evaluation strategy is adopted to screen target single plants in the BC2F1 population. That is, in the preliminary screening stage, 3 - 4 SNP reduced molecular markers corresponding to each chromosome are used to identify the corresponding exogenous target chromosomes respectively (except for 4 SNP loci on chromosome 1, and 3 SNP loci on other chromosomes), and the grains with complete introduction of exogenous target chromosomes are screened out. In the precise screening stage, the screened grains are sown. After 30 days of emergence, the leaf genomic DNA of each BC2F1 single plant is extracted, and genotyping is carried out using the Maize6H - 60K SNP genotyping array. The method in step (2) is used to evaluate the coverage rate of exogenous target chromosomes and the background recovery rate of non - target chromosomes of BC2F1 single plants. On the premise of the complete introduction of exogenous target chromosomes, the BC2F1 single plants are ranked from high to low according to the background recovery rate of non - target chromosomes. Finally, for each chromosome BC2F1 population, the top 5 BC2F1 single plants carrying exogenous target chromosomes and with the highest background recovery rate of non - target chromosomes are screened out and self - crossed to preserve the germplasm. This application provides the SNP locus information and its marker primer sequences required for preliminary screening in Table 1. Since the SNP reduced molecular markers in this application select the KASP molecular detection platform in the preliminary screening stage, the PCR amplification reaction system and reaction conditions of the KASP platform are provided in Tables 2 and 3 respectively.

[0059] (4) Using Jing 92 as the female parent and the top 1 BC2F1 single plants of 10 different chromosomes screened in step (3) as the male parents respectively, backcrossing is carried out during the flowering period to obtain 10 BC3F1 populations of different chromosomes. The endosperm and leaf genomic DNA of BC3F1 single plants are extracted by the magnetic bead method. The SNP reduced molecular markers and the Maize6H - 60K SNP genotyping array hierarchical evaluation strategy in step (3) are used to evaluate the coverage rate of exogenous target chromosomes and the background recovery rate of non - target chromosomes of BC3F1 single plants. On the premise of the complete introduction of exogenous target chromosomes, the BC3F1 single plants are ranked from high to low according to the background recovery rate of non - target chromosomes. Finally, for each chromosome BC3F1 population, the top 5 BC3F1 single plants carrying exogenous target chromosomes and with the highest background recovery rate of non - target chromosomes are screened out and self - crossed to obtain the BC3F2 population.

[0060] (5) Twenty days after the emergence of the BC3F2 population, genomic DNA was extracted from leaves using the magnetic bead method. The exogenous target chromosome coverage rate and the non-target chromosome background recovery rate of individual BC3F2 plants were evaluated using SNP simplified molecular markers and the Maize6H-60K SNP chip grading evaluation strategy. On the premise of the complete introduction of exogenous target chromosomes, the non-target chromosome background recovery rates of individual BC3F2 plants were ranked from high to low, and single chromosome substitution lines for each chromosome of Jing 92 were finally determined in the BC3F2 population.

[0061] (6)The BC3F2 target individual plants were planted in the field and self-crossed for 1-2 rounds during the flowering period to obtain homozygous single chromosome substitution lines. In this application, an SCSL population containing 10 Jing 92 single chromosome substitution lines SCSL01-SCSL10 was selected, and Figure 1 a genotype pattern diagram of each SCSL was provided.

[0062] Figure 2 A typical phenotypic difference was provided, that is, the anthocyanin color development at the base of the lemma of the tassel anthers of SCSL02 was dark red, the anthocyanin color development at the base of the lemma of the anthers of Jing 92 was colorless, and its F1 progeny also showed red lemma bases, indicating that there was a dominant gene locus regulating anthocyanin accumulation at the base of the lemma of the anthers on chromosome 2.

[0063] The method for extracting genomic DNA by the magnetic bead method was as follows:

[0064] (1)Collect endosperm or leaf tissues in a clean deep well plate, add 2 steel balls with a diameter of 2 mm into the deep well plate, and transfer it to liquid nitrogen for freezing. Use a tissue grinder (model SPEX Geno2010-230, origin: USA) to break the tissue samples. The endosperm does not require liquid nitrogen freezing treatment.

[0065] (2)Add 400 µL of 2% SDS lysis buffer (preheated at 75 °C in advance) into the sample well, lyse at 75 °C for 20 min, and mix well every 10 min.

[0066] (3)Add 134 µL of potassium acetate and mix well by shaking for 2 min.

[0067] (4)After covering the film, centrifuge at 3800 rpm for 10 min, and pipette 200 µL of the supernatant into a new 96-well deep well plate.

[0068] (5)Prepare a magnetic bead working solution according to the ratio of magnetic beads to isopropanol of 1:50. Add 200 µL of the magnetic bead working solution into each sample well, pipette and mix well for 2 min, centrifuge at 3800 rpm for 2 min, and place it on a magnetic rack.

[0069] After the magnetic beads are adsorbed by the magnetic rack, pour off the waste liquid, add 300 µL of Wash Ⅰ working solution (composition: 0.5 mol / L EDTA, 5 mol / L NaCl, 1 mol / L Tris-HCl, absolute ethanol), mix well for 2 min, and place on the magnetic rack.

[0070] After the magnetic beads are adsorbed by the magnetic rack, pour off the waste liquid, add 300 µL of Wash Ⅱ working solution (composition: 75% ethanol), mix well for 2 min, and place on the magnetic rack.

[0071] After the magnetic beads are adsorbed by the magnetic rack, pour off the waste liquid, air-dry at room temperature for 10 min, add 200 µL of ddH2O, shake and mix well, let stand for 5 min. After the magnetic beads naturally sink to the bottom, aspirate 150 µL of the DNA solution into a new 96-well PCR plate.

[0072] Use a Nanodrop 2000 spectrophotometer (Thermo Fisher, origin: USA) to measure the DNA concentration and quality, and dilute the DNA concentration to 20 ng / µL for standby.

[0073] Calculation methods for the coverage rate of the target chromosome and the background recovery rate of non-target chromosomes in the backcross population:

[0074] (1) In the BC generation, based on the gene chip data, screen for polymorphic SNP sites between the two parents and use them as tracking sites for subsequent evaluation. Using the polymorphic sites between the two parents, compare each individual plant in the BC population with the genotype of the recipient parent one by one, so that the genotype of each individual plant in the BC population is transformed into a digital code to represent the difference in genotype at each locus from the recipient parent. The specific transformation rules are as follows: 1) The genotype that is homozygous and consistent with the recipient parent is transformed into "1"; 2) The genotype that is heterozygous and corresponding to the two parents is transformed into "2"; 3) The genotype that is homozygous and consistent with the donor parent is transformed into "3"; 4) The missing genotype is transformed into "0".

[0075] (2) Based on the data noise reduction algorithm of the sliding window (Patent No.: ZL202210144099.7), perform data noise reduction on the comparison results of each individual plant in the BC population, and transform the digital code genotype into a block-based HTP molecular marker. Assign a digital code to the HTP block according to the digital coding type of the SNP sites within the HTP block, that is, if the proportion of a certain digital code within the HTP block ≥ 80%, then the HTP block is assigned this digital code, so as to reflect the difference between the individual plant in the population and the recipient parent on the HTP marker.

[0076] (3) Based on the data noise reduction algorithm of the sliding window, perform data noise reduction on the HTP results, and calculate the coverage rate of the exogenous target chromosome and the genetic background recovery rate of non-target chromosomes for each individual plant in the BC population. The calculation formulas are as follows:

[0077] Target chromosome coverage rate (%) = (Number of HTPs with the same digital coding type as the donor parent HTP) / (Total number of tracked HTPs) × 100%

[0078] Non-target chromosome background recovery rate (%) = (Number of HTPs with the same digital coding type as the recipient parent HTP) / (Total number of tracked HTPs) × 100%

[0079] Note: The coverage rate and recovery rate of single chromosomes and the whole genome are calculated according to this method.

[0080] Table 1. 31 SNP loci and their KASP primer information

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Table 2. PCR reaction system based on the KASP platform

[0087]

[0088] Table 3. PCR reaction program based on the KASP platform

[0089]

[0090] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A set of SNP molecular markers for efficient breeding of maize single chromosome substitution lines, characterized in that: The SNP molecular marker is selected from any one or more of the following: Chr 1:11486898(B73_v3)A / G, Chr 1:35047742(B73_v3)A / G, Chr 1:156554418(B73_v3)G / T, Chr 1:290379031(B73_v3)A / G, Chr 2:1283337(B73_v3)C / T, Chr 2:109563977(B73_v3)G / T, Chr 2:223977329(B73_v3)G / T, Chr 3:15222626(B73_v3)G / T, Chr 3:88400490(B73_v3)A / G、Chr 3:212050056(B73_v3)A / C, Chr 4:18832692(B73_v3)A / C, Chr4:128874467(B73_v3)G / T, Chr 4:240891846(B73_v3)A / C, Chr 5:6881508(B73_v3)C / T, Chr 5:88226331(B73_v3)C / T, Chr 5:204879477(B73_v3)A / G, Chr 6:4843862(B73_v3)T / C, Chr 6:39822980(B73_v3)T / C、Chr 6:158855834(B73_v3)A / G, Chr 7:13214477(B73_v3)A / C, Chr 7:88150665(B73_v3)G / T, Chr 7:175252106(B73_v3)A / G, Chr 8:7624060(B73_v3)G / T, Chr 8:59490913(B73_v3)A / G, Chr 8:165927215(B73_v3)T / G, Chr 9:11505101(B73_v3)C / T, Chr 9:61772044(B73_v3)C / T、Chr 9:155054805(B73_v3)A / G, Chr 10:2802839(B73_v3)A / C, Chr 10:39960290(B73_v3)C / T, Chr 10:130399836(B73_v3)A / G.

2. A primer for detecting the SNP molecular marker according to claim 1, characterized in that: The sequence of the primer is shown in any one or more of SEQ ID NO: 1-SEQ ID NO:

93.

3. A detection product comprising the primer according to claim 2; Preferably, the detection product comprises a detection reagent or a kit.

4. Use of the SNP molecular marker according to claim 1, the primer according to claim 2 or the detection product according to claim 3 in the breeding of maize single chromosome substitution lines.

5. A method for efficiently breeding single chromosome substitution lines in maize, characterized in that: The method comprises: using the SNP molecular marker of claim 1, the primer of claim 2 or the detection product of claim 3 to perform preliminary screening of target individual plants; Preferably, the method further comprises using a gene chip for genotyping, using HTP molecular markers to perform foreground and background selection on the initially screened backcross progeny, and screening individuals with high foreground coverage and background recovery rates; Preferably, the method uses Jing 724 as the donor parent and Jing 92 as the recipient parent.

6. The method according to claim 5, characterized in that The method for selecting foreground and background of backcross progeny includes: using gene chips for genotyping, comparing the genotypes of the backcross progeny with those of the recipient parent, converting them into block HTP molecular markers, and calculating the foreground coverage and background recovery rate of the backcross progeny; Preferably, the gene chip is a Maize6H-60K SNP gene chip; Preferably, the method for performing foreground and background selection on backcross progeny further comprises: performing data noise reduction before and after the genotype is converted into an HTP molecular marker; Preferably, data denoising is performed based on a sliding window data denoising algorithm; Preferably, the foreground coverage and background recovery rate include the exogenous target chromosome coverage rate and the non-target chromosome genetic background recovery rate; Preferably, the screening condition for the target individual plant is that the backcross progeny have a high coverage rate of the exogenous target chromosome and a top 5 background recovery rate of the non-target chromosome.

7. The method according to claim 5 or 6, characterized in that: The specific steps of the method include: (1) Using the maize inbred line Jing 92 as the recipient parent and Jing 724 as the donor parent, a hybrid was performed to obtain the F1 offspring, and using Jing 92 as the female parent and the F1 offspring as the male parent, a BC1F1 backcross offspring was obtained; (2) Using the polymorphic SNP sites identified by gene chip genotyping between the parents, the genotypes of the BC1F1 strain were compared with those of the recipient parent and converted into block-based HTP molecular markers to calculate the foreground coverage and background recovery rate of the BC1F1 strain; (3) Using Jing 92 as the female parent and the BC1F1 plant selected in step (2) as the male parent, backcrossing is performed to obtain BC2F1 backcross progeny, and the SNP molecular markers described in claim 1 and the Maize6H-60K SNP gene chip are used for screening to calculate the foreground coverage rate and background recovery rate of the BC2F1 plant; (4) Using Jing 92 as the female parent and the BC2F1 plant selected in step (3) as the male parent, backcrossing is performed to obtain BC3F1 backcross progeny, and the SNP molecular markers described in claim 1 and the Maize6H-60K SNP gene chip are used for screening to calculate the foreground coverage rate and background recovery rate of the BC3F1 plant, and the screened BC3F1 plant is self-pollinated to obtain BC3F2 progeny; (5) Using the SNP molecular markers described in claim 1 and the Maize6H-60K SNP gene chip for screening, the foreground coverage and background recovery rate of the BC3F2 single plant were calculated, and the single chromosome substitution line of each chromosome of Jing 92 was determined respectively; (6) BC3F2 individual plants were self-pollinated to obtain homozygous single chromosome substitution lines.

8. Use of the SNP molecular marker according to claim 1, the primer according to claim 2, the detection product according to claim 3 or the single chromosome substitution line obtained by the method according to any one of claims 5 to 7 in maize genetic research.

9. Use of the SNP molecular marker according to claim 1, the primer according to claim 2, the detection product according to claim 3 or the single chromosome substitution line obtained by the method according to any one of claims 5 to 7 in corn genetic breeding.

10. Use of the SNP molecular marker according to claim 1, the primer according to claim 2, the detection product according to claim 3 or the single chromosome substitution line obtained by the method according to any one of claims 5 to 7 in the creation of new maize F2, DH, and CSSL populations.

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