High efficient breeding method of maize monosomic substitution line and its application

Abstract

The application discloses a high-efficiency breeding method of a corn single chromosome substitution line and application thereof. The application provides a Single Chromosome Substitution Line (SCSL) breeding method which is rapid, efficient, precise and high-throughput by using SNP and HTP type molecular markers and adopting a simplified marker-gene chip hierarchical evaluation strategy. Taking the male inbred line Jing 92 and the female inbred line Jing 724 of the main promoted corn variety Jingke 968 as parent materials, a first corn SCSL population is successfully created. Through implementation of the method, the problems of low efficiency, large resource consumption, complex genetic background, and non-compatibility of genetic positioning and breeding in traditional genetic population creation are solved, a new population creation method which is efficient, economical, single in genetic background and compatible with genetic positioning and breeding is provided, and new scheme support is provided for corn genetic research and precise breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant breeding, and particularly relates to a high-efficiency breeding method for a maize single-chromosome substitution line and application thereof. BACKGROUND

[0002] Maize is the most widely planted crop for food and feed in the world, and is also a typical model crop in genetic breeding research. The creation of maize genetic populations provides an important research basis for the mining 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 selected as parents, and after years of hybridization, backcross (BC) and selfing, 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] At present, simple sequence repeats (SSR) are mostly used in the creation of maize CSSL, and the marker number flux and sample detection flux are low, which easily causes the loss of target single individuals. At the same time, as a genetic population, the CSSL cannot achieve whole genome coverage of the exogenous fragment during the creation process, so as many individuals as possible that meet the requirements need to be retained to ensure the whole genome coverage of the exogenous chromosomal fragment. Most CSSLs can only be used for gene positioning of specific different traits, cannot simultaneously position multiple traits, and cannot be used for maize genetic breeding. Therefore, it is urgent to create a genetic population that takes into account positioning and breeding, and to develop a set of rapid, efficient, accurate and high-throughput breeding methods for chromosomal substitution lines as a new tool for maize genetic basic research and variety improvement, to ensure the development and innovation of maize breeding. SUMMARY

[0004] In order to make up for the deficiencies of the prior art, the present application provides a high-efficiency breeding method for a maize single-chromosome substitution line and application thereof.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The first aspect of the present application provides a set of SNP molecular markers for high-efficiency breeding of maize single-chromosome substitution lines, wherein 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 application provides a primer for detecting the SNP molecular marker of the first aspect of the present application, and the sequence of the primer is shown in any one or more of SEQ ID NO: 1-SEQ ID NO: 93.

[0009] The third aspect of the present application provides a detection product comprising the primer of the second aspect of the present application.

[0010] Further, the detection product comprises a detection reagent or a kit.

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

[0012] The fifth aspect of the present application provides a high-efficiency breeding method of maize single chromosome substitution line, which comprises: using the SNP molecular marker of the first aspect of the present application, the primer of the second aspect of the present application or the detection product of the third aspect of the present application to perform preliminary screening of the target single plant.

[0013] Further, the method further comprises using a gene chip to perform genotyping, using HTP molecular markers to perform foreground and background selection on the backcross progeny screened preliminarily, and screening individuals with high foreground coverage and background recovery rate.

[0014] Further, the method takes Jing724 as the donor parent and Jing92 as the recipient parent.

[0015] Further, the method for performing foreground and background selection on the backcross progeny comprises: performing genotyping by using a gene chip, comparing the genotypes of the backcross progeny and the recipient parent, and converting the genotypes into block HTP molecular markers, and calculating the foreground coverage and the background recovery rate of the backcross progeny.

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

[0017] Further, the method for performing foreground and background selection on the backcross progeny further comprises: performing data denoising before and after the genotypes are converted into HTP molecular markers.

[0018] Further, the data denoising is performed by using a sliding window-based data denoising algorithm.

[0019] Further, the foreground coverage and the background recovery rate comprise the coverage rate of the exogenous target chromosome and the genetic background recovery rate of the non-target chromosome.

[0020] Further, the screening condition of the target single plant is the backcross progeny with high coverage rate of the exogenous target chromosome and top 5 background recovery rate of the non-target chromosome.

[0021] Further, the specific steps of the method comprise:

[0022] (1) taking the maize inbred line Jing92 as the recipient parent and Jing724 as the donor parent, performing hybridization, taking Jing92 as the female parent and the F1 progeny as the male parent, and obtaining the BC1F1 backcross progeny;

[0023] (2) The genotype of the BC1F1 single plant and the genotype of the receptor parent are compared by using the polymorphic SNP site identified by the Maize 6H-60K SNP gene chip genotyping, and are converted into a block HTP molecular marker, and the foreground coverage and background recovery rate of the BC1F1 single plant are calculated;

[0024] (3) The BC2F1 backcross offspring is obtained by backcrossing Jing 92 as the female parent and the BC1F1 single plant screened in step (2) as the male parent, and the SNP molecular marker and the Maize 6H-60K SNP gene chip are used for screening, and the foreground coverage and background recovery rate of the BC2F1 single plant are calculated;

[0025] (4) The BC3F1 backcross offspring is obtained by backcrossing Jing 92 as the female parent and the BC2F1 single plant screened in step (3) as the male parent, and the SNP molecular marker and the Maize 6H-60K SNP gene chip are used for screening, and the foreground coverage and background recovery rate of the BC3F1 single plant are calculated, and the screened BC3F1 single plant is selfed to obtain BC3F2 offspring;

[0026] (5) The SNP molecular marker and the Maize 6H-60K SNP gene chip are used for screening, and the foreground coverage and background recovery rate of the BC3F2 single plant are calculated, and the single chromosome substitution line of Jing 92 on each chromosome is determined.

[0027] (6) The single chromosome substitution line is obtained by selfing the BC3F2 single plant.

[0028] The sixth aspect of the present application provides an application of the single chromosome substitution line obtained by the SNP molecular marker of the first aspect of the present application, the primer of the second aspect of the present application, the detection product of the third aspect of the present application or the method of the fifth aspect of the present application in corn genetics research.

[0029] The seventh aspect of the present application provides an application of the single chromosome substitution line obtained by the SNP molecular marker of the first aspect of the present application, the primer of the second aspect of the present application, the detection product of the third aspect of the present application or the method of the fifth aspect of the present application in corn genetic breeding.

[0030] The eighth aspect of the present application provides an application of the single chromosome substitution line obtained by the SNP molecular marker of the first aspect of the present application, the primer of the second aspect of the present application, the detection product of the third aspect of the present application or the method of the fifth aspect of the present application in the creation of corn F2, DH, CSSL new population.

[0031] The advantages and beneficial effects of the present application are as follows:

[0032] The application provides a single chromosome substitution line (SCSL) breeding method using SNP and HTP type molecular markers, adopting a "simplified marker-gene chip" hierarchical evaluation strategy, which is fast, efficient, accurate and high-throughput. Taking the male inbred line Jing 92 and the female inbred line Jing 724 of the main recommended variety Jingke 968 as parent materials, the first SCSL population of corn is successfully created. Compared with the prior art, the application has the following advantages:

[0033] 1. Efficient and accurate screening of target single plant: In the application, the BC population of each generation is evaluated for genetic background recovery rate and target foreign chromosome coverage rate by combining high-density and high-throughput SNP gene chip with high-throughput SNP simplified molecular marker of sample. A small number of SNP simplified molecular markers are developed using the polymorphic SNP sites between the parents of the BC population. The genotype of the seed endosperm or leaf of a large number of BC offspring individuals is detected by using SNP simplified markers, and individuals with higher coverage of target foreign chromosomes are quickly screened out. The small-scale BC individuals screened out are subjected to Maize 6H-60K SNP gene chip genotyping and converted into HTP molecular markers to accurately evaluate the target chromosome coverage rate and the background recovery rate of non-target chromosomes. Finally, individuals meeting the breeding requirements are determined, thereby improving the accuracy of target single plant screening.

[0034] 2. Precise background evaluation algorithm: In the application, the data denoising algorithm based on sliding window fully utilizes the strong linkage of SNP sites within HTP markers, converts SNP molecular markers into block HTP molecular markers, and accurately evaluates the target chromosome coverage rate and the background recovery rate of non-target chromosomes of BC population individuals through HTP molecular markers.

[0035] 3. Significantly reduce the cost of population breeding: The population size planted in the field is greatly reduced by SNP gene chip and simplified marker evaluation and screening, and only the target single plant screened by the gene chip needs to be backcrossed and selfed, thereby greatly reducing the field planting area and pollination amount and improving the resource utilization rate in the creation process. The "simplified marker-gene chip" hierarchical evaluation strategy is adopted, i.e., a small number of SNP simplified markers with high-throughput sample and low-cost genotyping are used to preliminarily screen a large number of BC populations, and only a small number of BC individuals screened preliminarily need to be accurately typed by high-density SNP gene chip, thereby realizing a cost-effective detection scheme, reducing the overall cost, and shortening the breeding cycle of target individuals.

[0036] By implementing the method of the present application, the problems of low efficiency, large resource consumption, complex genetic background, and incompatibility of genetic positioning and breeding of traditional genetic populations are solved, a new population creation method that is efficient, economical, single background, and compatible with genetic positioning and breeding is provided, and a new scheme support is provided for corn genetic research and precision breeding. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a BC3F2 genotype pattern diagram of 10 Jing92 single chromosome substitution (Maize 6H-60K);

[0038] Figure 2 is an anther and glume base anthocyanin color identification diagram of corn single chromosome substitution line SCSL02 and inbred parent Jing92. DETAILED DESCRIPTION

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

[0040] The present application provides a set of SNP molecular markers for efficient breeding of corn single chromosome substitution lines.

[0041] In some embodiments, SNP refers to Single Nucleotide Polymorphism, mainly refers to DNA sequence polymorphism caused by single nucleotide variation at the genomic level, and the single nucleotide variation includes variation caused by single base transition, transversion, insertion or deletion.

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

[0043] In some embodiments, the primers are KASP (Kompetitive Allele Specific PCR) primers, which can or can 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 attached to a nucleic acid. Labels include, but are not limited to, fluorescent groups such as FAM, HEX; dyes; radioactive labels such as 32P; binding moieties such as biotin; hapten such as digoxigenin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The label can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The label can include or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly without a label (e.g., the sequence is read directly).

[0044] In preferred embodiments, the label is selected from a fluorescent group.

[0045] In particular embodiments, the fluorescent group is selected from FAM, HEX.

[0046] The present application provides a detection product comprising the above-mentioned primers.

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

[0048] In some embodiments, the kit further comprises deionized water, a buffer, a preservative, or a protein stabilizer. The kit can also contain the necessary components for detecting the detection reagent (e.g., a substrate, an enzyme, etc.). The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample contained. Each component of the kit is typically packaged in a separate container, all of the different containers are packed in one package with instructions containing the composition of the product, the way of storage, etc.

[0049] The components of a kit can be packaged in the form of an aqueous medium or in the form of a lyophilized material. Suitable containers for the components of a kit will typically include at least one vial, test tube, flask, bottle, syringe or other container, into which a component can be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the various components can be present in a shared container or separate containers. A kit of the application will also typically include a container into which a reagent can be contained, sealed for commercial sale. Such container can include an injection or blow molded plastic container into which the desired vial can be retained.

[0050] The solid support of the kit can be, for example, plastic, silicon chip, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide. The biological sample can be, for example, animal or plant cell culture, cell line, tissue, organ, etc.

[0051] In some embodiments, in the process of SCSL creation, the introduction of the exogenous target chromosome provided by the donor parent in the population is identified by molecular marker technology, and the process of screening the target single plant carrying the exogenous target chromosome is called foreground selection; the foreground coverage refers to the proportion of the offspring individuals carrying the exogenous target chromosome fragment. The process of identifying the recovery of the genetic background of the recipient parent in the population by molecular marker technology is called background selection; the background recovery rate refers to the recovery degree of the genetic background of the recipient parent in the offspring individuals, that is, the proportion of the genotype of the non-target chromosome consistent with the genotype of the recipient parent.

[0052] In some embodiments, the corn SCSL of the application is used as a genetic population for functional research such as gene mapping. By comparing the differences between SCSL01-SCSL10 and the parental inbred lines Jing92 and Jing724 in the field, the specific chromosome where the gene locus associated with the difference phenotype is located can be quickly determined. The application provides a typical trait difference phenotype in the Figure 2 SCSL02, that is, the anther and lemma base anthocyanin of SCSL02 is deep red, the anther and lemma base anthocyanin of Jing92 is colorless, and the F1 offspring also shows red lemma base, indicating that there is a dominant gene locus on chromosome 2 that regulates the accumulation of anther and lemma base anthocyanin. It can also be used as a "bridge parent" for genetic improvement such as corn excellent gene aggregation. It can also be used as a basic material, which can be used for the creation of new populations such as DH and CSSL through techniques such as double haploid (DH).

[0053] The application will be further described in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and not as limitations to the present application. The principal features of the present application can be employed in various embodiments without departing from the scope of the present application.

[0054] Examples

[0055] The process of creating the corn SCSL population of the present application is as follows:

[0056] (1) Using corn inbred line Jing 92 as the recipient parent (female parent) and Jing 724 as the donor parent (male parent), F1 progeny were obtained by crossing at the flowering stage. Using Jing 92 as the female parent and F1 progeny as the male parent, BC1F1 generation was obtained by backcrossing at the flowering stage. Subsequently, Maize 6H-60K SNP gene chip was used to evaluate the foreign target chromosome coverage and non-target chromosome background recovery rate of BC1F1 single plants.

[0057] (2) The genomic DNA of Jing 92, Jing 724 and BC1F1 single plants was extracted by magnetic bead method, and genotyping was performed using Maize 6H-60K SNP gene chip. Based on the gene chip data, SNP sites with polymorphic differences between Jing 92 and Jing 724 were screened. Using the polymorphic SNP sites between the parents, the genotypes of BC1F1 single plants were compared with those of the recipient parent, so that the genotypes of BC1F1 single plants were converted into digital codes. Based on the data denoising algorithm of sliding window, the genotype comparison results of 10 chromosomes of BC1F1 single plants were denoised, and converted into block HTP molecular markers. Based on the data denoising algorithm of sliding window, the HTP results were denoised, and the foreign target chromosome coverage and non-target chromosome genetic background recovery rate of BC1F1 single plants were calculated. Under the premise of complete introduction of foreign target chromosomes, the non-target chromosome background recovery rate of BC1F1 single plants was sorted from high to low, and finally the BC1F1 single plants with foreign target chromosome coverage (foreground coverage) close to 100% and non-target chromosome background recovery rate (background recovery rate) top 5 were selected for each chromosome.

[0058] (3) Taking Jing 92 as the female parent, the top 1 BC1F1 single plant of 10 different chromosomes screened out in step (2) is taken as the male parent, and backcross is carried out at the flowering stage to obtain 10 different chromosome BC2F1 populations. The endosperm genomic DNA of the BC2F1 single plant is extracted by the magnetic bead method. The target single plant is screened out in the BC2F1 population by using the "simplified marker-gene chip" hierarchical evaluation strategy. That is, in the preliminary screening stage, 3-4 SNP simplified molecular markers corresponding to each chromosome are used to identify the corresponding exogenous target chromosome (except that 1 chromosome has 4 SNP sites, and other chromosomes have 3 SNP sites), and the grain with complete introduction of the exogenous target chromosome is screened out. In the precise screening stage, the screened grain is sown, and after 30 days of seedling, the leaf genomic DNA of each BC2F1 single plant is extracted, and genotyping is carried out by using Maize6H-60K SNP gene chip. The exogenous target chromosome coverage and non-target chromosome background recovery rate of the BC2F1 single plant are evaluated by the method of step (2). On the premise of complete introduction of the exogenous target chromosome, the non-target chromosome background recovery rate of the BC2F1 single plant is ranked from high to low, and finally the BC2F1 single plant carrying the exogenous target chromosome and having the top 5 non-target chromosome background recovery rate of each chromosome BC2F1 population is screened out, and is self-crossed to reserve the germplasm. The SNP site information and marker primer sequences required for preliminary screening are provided in Table 1. Since the SNP simplified molecular marker in the present application selects 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) Taking Jing 92 as the female parent, the top 1 BC2F1 single plant of 10 different chromosomes screened out in step (3) is taken as the male parent, and backcross is carried out at the flowering stage to obtain 10 different chromosome BC3F1 populations. The endosperm and leaf genomic DNA of the BC3F1 single plant is extracted by the magnetic bead method. The exogenous target chromosome coverage and non-target chromosome background recovery rate of the BC3F1 single plant are evaluated by using the SNP simplified molecular marker and Maize6H-60K SNP chip hierarchical evaluation strategy of step (3). On the premise of complete introduction of the exogenous target chromosome, the non-target chromosome background recovery rate of the BC3F1 single plant is ranked from high to low, and finally the BC3F1 single plant carrying the exogenous target chromosome and having the top 5 non-target chromosome background recovery rate of each chromosome BC3F1 population is screened out, and is self-crossed to obtain the BC3F2 population.

[0060] (5) After 20 days of emergence, the genomic DNA of the leaves was extracted by magnetic bead method, and the coverage of the exogenous target chromosome and the background recovery rate of the non-target chromosome of the BC3F2 single plant were evaluated by using the SNP simple molecular marker and the Maize6H-60K SNP chip hierarchical evaluation strategy. On the premise of complete introduction of the exogenous target chromosome, the background recovery rate of the non-target chromosome of the BC3F2 single plant was sorted from high to low, and finally the monosomic substitution line of each chromosome of Jing 92 was determined in the BC3F2 population.

[0061] (6) The target single plant of BC3F2 was planted in the field, and 1-2 rounds of selfing were performed at the flowering stage to obtain the homozygous monosomic substitution line. The SCSL population containing 10 Jing 92 monosomic substitution lines SCSL01-SCSL10 was bred by the present application, and the genotype pattern of each SCSL was provided in Figure 1 .

[0062] Figure 2 A typical trait difference phenotype was provided, i.e., the anther glume base anthocyanin of SCSL02 was colored as dark red, the anther glume base anthocyanin of Jing 92 was colorless, and the F1 offspring also showed red glume base, indicating that there was a dominant gene site on chromosome 2 that regulated the accumulation of anther glume base anthocyanin.

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

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

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

[0066] (3) Add 134 μL of potassium acetate, shake and mix for 2 min.

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

[0068] (5) Prepare the magnetic bead working solution according to the ratio of 1:50 of magnetic beads and isopropyl alcohol, add 200 μL of magnetic bead working solution to each sample well, mix by aspiration and beating for 2 min, centrifuge at 3800 rpm for 2 min, and place on the magnetic stand.

[0069] (6) After the magnetic beads were adsorbed by the magnetic stand, the waste liquid was poured out, 300 μL of Wash I working solution (composition: 0.5 mol / L EDTA, 5 mol / L NaCl, 1 mol / L Tris-HCl, anhydrous ethanol) was added, mixed for 2 min, and placed on the magnetic stand.

[0070] (7) After the magnetic beads were adsorbed by the magnetic stand, the waste liquid was poured out, 300 μL of Wash II working solution (composition: 75% ethanol) was added, mixed for 2 min, and placed on the magnetic stand.

[0071] (8) After the magnetic beads were adsorbed by the magnetic stand, the waste liquid was poured out, and the room temperature was left for 10 min. 200 μL of ddH2O was added, mixed by shaking, and left for 5 min. After the magnetic beads naturally settled to the bottom, 150 μL of DNA solution was taken to a new 96-well PCR plate.

[0072] (9) The DNA concentration and quality were determined using a Nanodrop 2000 spectrophotometer (Thermo, USA), and the DNA concentration was diluted to 20 ng / μL for standby.

[0073] Calculation method of target chromosome coverage and non-target chromosome background recovery rate of backcross population:

[0074] (1) In the BC generation, based on the SNP sites with polymorphic differences between the parents, they were selected as tracking sites for subsequent evaluation. Using the polymorphic sites between the parents, the BC population was compared with the genotype of the recipient parent one by one, so that the genotype of the BC population was converted to digital coding to represent the difference between the genotype at each site and the recipient parent. The specific conversion rules are as follows: 1) the genotype of homozygous and consistent with the recipient parent is converted to "1"; 2) the genotype of heterozygous and corresponding to the parents is converted to "2"; 3) the genotype of homozygous and consistent with the donor parent is converted to "3"; 4) the missing genotype is converted to "0".

[0075] (2) The data denoising algorithm based on sliding window (patent number: ZL202210144099.7) was used to denoise the comparison results of the BC population, and the digital coding genotype was converted to block HTP molecular markers. According to the digital coding type of the SNP sites in the HTP block, the HTP block was assigned a digital code, i.e. if the proportion of a certain digital code in the HTP block is ≥80%, the HTP block is assigned the digital code, thereby reflecting the difference between the population and the recipient parent at the HTP marker.

[0076] (3) The data denoising algorithm based on sliding window was used to denoise the HTP results, and the exogenous target chromosome coverage and non-target chromosome genetic background recovery rate of the BC population were calculated. The calculation formula is as follows:

[0077] Target chromosome coverage (%) = (number of HTPs identical to the donor parent HTP digital coding type) / (total number of tracked HTPs) x 100%

[0078] Non-target chromosome background recovery rate (%) = (number of HTPs identical to the recipient parent HTP digital coding type) / (total number of tracked HTPs) x 100%

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

[0080] Table 131 SNP sites and KASP primer information

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Table 2 KASP platform-based PCR reaction system

[0087]

[0088] Table 3 KASP platform-based PCR reaction program

[0089]

[0090] The above examples are only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A primer set, characterized by, The sequence of the primer set consists of SEQ ID NO: 1-SEQ ID NO:

93.

2. A detection product comprising the primer set of claim 1.

3. The test product according to claim 2, characterized in that The detection product comprises a detection reagent or a kit.

4. Use of the primer set of claim 1 or the detection product of any one of claims 2-3 in breeding of a maize single chromosome substitution line, wherein the maize single chromosome substitution line is constructed by taking Jing 724 as a donor parent and Jing 92 as a recipient parent.

5. A high efficiency selection method of a maize monochromosome substitution line, characterized in that, The method comprises: performing preliminary screening of target single plants by using the primer set of claim 1 or the detection product of any one of claims 2-3, and taking Jing 724 as a donor parent and Jing 92 as a recipient parent.

6. The method of claim 5, wherein, The method further comprises: performing genotyping by using a gene chip, performing foreground and background selection of the preliminary screened backcross progeny by using HTP molecular markers, and screening individuals with high foreground coverage and background recovery rate.

7. The method of claim 6, wherein, The method for performing foreground and background selection on the backcross progeny comprises: performing genotyping by using a gene chip, comparing the genotypes of the backcross progeny and the recipient parent, and converting the genotypes into block HTP molecular markers, and calculating the foreground coverage and the background recovery rate of the backcross progeny.

8. The method of claim 7, wherein, The gene chip is a Maize6H-60K SNP gene chip.

9. The method of claim 7, wherein, The method for performing foreground and background selection on the backcross progeny further comprises: performing data denoising before and after the genotypes are converted into HTP molecular markers.

10. The method of claim 9, wherein, The data denoising is performed by using a sliding window-based data denoising algorithm.

11. The method of claim 7, wherein, The foreground coverage and the background recovery rate comprise exogenous target chromosome coverage and non-target chromosome genetic background recovery rate.

12. The method of claim 5, wherein, The screening condition of the target single plant is a backcross progeny with high exogenous target chromosome coverage and top 5 non-target chromosome background recovery rate.

13. The method according to any one of claims 5-12, characterized in that, The specific steps of the method comprise: (1) taking a maize inbred line Jing 92 as a recipient parent and Jing 724 as a donor parent to perform hybridization, taking Jing 92 as a female parent and the F1 progeny as a male parent to obtain BC1F1 backcross progeny; (2) comparing the genotypes of the BC1F1 single plant and the recipient parent by using the polymorphic SNP sites identified by parent gene chip genotyping, converting the genotypes into block HTP molecular markers, and calculating the foreground coverage and the background recovery rate of the BC1F1 single plant; (3) taking Jing 92 as a female parent and the BC1F1 single plant screened in step (2) as a male parent to perform backcrossing, obtaining BC2F1 backcross progeny, and performing screening by using the primer set of claim 1 and the Maize6H-60K SNP gene chip to calculate the foreground coverage and the background recovery rate of the BC2F1 single plant; (4) taking Jing 92 as a female parent and the BC2F1 single plant screened in step (3) as a male parent to perform backcrossing, obtaining BC3F1 backcross progeny, performing screening by using the primer set of claim 1 and the Maize6H-60K SNP gene chip to calculate the foreground coverage and the background recovery rate of the BC3F1 single plant, and self-crossing the screened BC3F1 single plant to obtain BC3F2 progeny; (5) using the primer set and the Maize 6H-60K SNP gene chip screening of claim 1 to calculate the foreground coverage rate and the background recovery rate of the BC3F2 single plant, and to determine the single chromosome substitution line of each chromosome of Jing 92 respectively; (6) selfing the BC3F2 single plant to obtain the homozygous single chromosome substitution line.

Images