InDel molecular marker combination for corn variety identification and application of InDel molecular marker combination

Abstract

The invention discloses an InDel molecular marker combination for maize variety identification and application of the InDel molecular marker combination, and belongs to the field of molecular biology. The InDel molecular marker is combined with 80 insertion / deletion fragments greater than or equal to 50 bp, and InDel loci which are uniformly distributed in 10 chromosomes and are rich in polymorphism. The InDel molecular marker combination has high genetic stability and can accurately distinguish related varieties including sister lines. The method gets rid of dependence on capillary electrophoresis or fluorescence detection equipment, the detection cost is reduced by 80% or above, and related varieties can be effectively distinguished. The method is easy to operate, visual in result and especially suitable for basic units.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an InDel molecular marker combination for maize variety identification and its application. Background Technology

[0002] In the field of crop variety identification, DNA fingerprinting technology has become an important tool for ensuring variety authenticity, maintaining seed market order, and protecting breeders' intellectual property rights. Currently, maize variety identification in my country mainly relies on the national standard "Detection of Authenticity and Purity of Major Crop Varieties by SSR Molecular Markers - Maize" (GB / T39914-2021), which uses SSR (Simple Sequence Repeat) markers for detection. However, SSR markers have significant limitations in practical applications: on the one hand, their detection relies on polyacrylamide gel electrophoresis or capillary electrophoresis platforms, which are cumbersome and require sophisticated equipment, resulting in high detection costs, making it difficult for many grassroots seed management departments and enterprises to conduct on their own; on the other hand, SSR markers may exhibit genetic instability at some heterozygous loci, especially when the parents do not reach homozygosity at specific loci, which may lead to inaccurate identification results or even termination of detection, affecting the comprehensiveness and reliability of variety identification.

[0003] To overcome the limitations of SSR markers, the development of next-generation sequencing technology has promoted the application of SNP (single nucleotide polymorphism) and InDel (insertion / deletion) markers in maize genome research. While SNP markers have the advantages of being numerous and widely distributed, their mainstream detection methods, such as KASP, still rely on fluorescence detection equipment, resulting in high costs and hindering widespread adoption in grassroots units with limited funding and technology. In contrast, InDel markers, with their obvious length polymorphism and direct interpretation via conventional PCR amplification and agarose gel electrophoresis, exhibit significant advantages in ease of operation and cost control. However, current technologies still lack a systematically screened and validated InDel molecular marker combination specifically for maize variety identification. This lack of a combination fails to meet the demand for low-threshold, low-cost applications while ensuring high accuracy, representing a pressing technical bottleneck in current grassroots seed supervision and market anti-counterfeiting efforts.

[0004] Therefore, developing a set of InDel molecular markers that are highly compatible, stable, and easy to promote, based on the existing SSR marker system, is of urgent practical significance for supplementing and improving the national standard testing system, enhancing the efficiency of local seed supervision, and reducing the cost of rights protection for enterprises and farmers. This requires that the selected markers not only possess good polymorphism and variety differentiation capabilities, but also be suitable for simple experimental conditions, thereby truly achieving technology dissemination and widespread application, and providing a reliable and practical new solution for maize variety identification. Summary of the Invention

[0005] The purpose of this invention is to provide an InDel molecular marker combination for maize variety identification and its application, thereby addressing the problems existing in the prior art. This invention screens 80 highly polymorphic InDel marker combinations, enabling accurate identification of maize varieties using conventional PCR and agarose gel electrophoresis. This invention eliminates reliance on capillary electrophoresis or fluorescence detection equipment, reducing detection costs by over 80%, and effectively distinguishes closely related varieties. It is simple to operate, provides intuitive results, and is particularly suitable for promotion in grassroots units.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an InDel molecular marker combination for maize germplasm / variety identification, characterized in that the InDel molecular marker combination consists of InDel sites located at B73 of the reference genome as shown in Table 1.

[0007] The present invention also provides a primer set for amplifying the InDel molecular marker combination, which consists of primers with nucleotide sequences as shown in SEQ ID NO.1-160.

[0008] The present invention also provides a kit comprising the said primer set.

[0009] This invention also provides the application of the InDel molecular marker combination, the primer set, or the kit described herein in constructing fingerprint profiles of maize varieties.

[0010] This invention also provides a method for constructing a fingerprint profile of a maize variety, comprising the following steps: (1) Extract genomic DNA from maize variety samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set or the kit described above; (3) Perform electrophoresis on the amplification products of step (2) and determine the genotype of each site of the maize variety in the InDel molecular marker combination described in the claims based on the position of the electrophoretic bands; (4) The genotype information obtained in step (3) is digitally recorded to form a specific fingerprint map of the maize variety to be tested.

[0011] The present invention also provides an application of the InDel molecular marker combination, the primer set, or the kit described herein in maize variety identification.

[0012] This invention also provides a method for identifying maize varieties, comprising the following steps: (1) Extract genomic DNA from the maize sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set or the kit described above; (3) Perform electrophoresis detection on the amplification products of step (2) and determine the genotype of each site in the InDel molecular marker combination described in the claims based on the position of the electrophoretic bands; (4) Compare the genotype obtained in step (3) with the fingerprint of known maize varieties, and determine the variety identity of the maize sample to be tested based on the comparison results.

[0013] The present invention also provides an application of the InDel molecular marker combination, the primer set, or the kit described herein in the detection of genetic kinship among maize varieties.

[0014] This invention also provides the application of the InDel molecular marker combination, the primer set, or the kit in maize germplasm resource analysis.

[0015] This invention also provides the application of the InDel molecular marker combination, the primer set, or the kit described herein in maize breeding population segmentation or variety uniformity identification.

[0016] The present invention discloses the following technical effects: This invention provides a set of InDel molecular markers specifically for maize variety identification and their applications, effectively solving the technical bottlenecks of high cost and strong equipment dependence of existing SSR marker detection, as well as the difficulty in popularizing SNP marker fluorescence detection platforms. This marker set, through a rigorous genomic screening strategy, obtained 80 polymorphic InDel loci with insertion / deletion fragments ≥50 bp, evenly distributed across 10 chromosomes, ensuring clear interpretation via conventional PCR combined with agarose gel electrophoresis. This significantly reduces the detection threshold and cost (single-sample testing costs are more than 80% lower than commissioned SSR testing), and the markers exhibit high genetic stability, accurately distinguishing closely related varieties, including sister lines.

[0017] Furthermore, this invention provides a complete set of amplification primers, reagent kits, and standardized detection and analysis methods, forming a complete technical solution from DNA extraction, PCR amplification, electrophoresis detection to fingerprint pattern construction and variety identification. This invention can not only be used for rapid identification of variety authenticity and purity, but also has wide applications in germplasm resource genetic diversity analysis, breeding population division, and variety rights protection, exhibiting good versatility and operability. The entire technology has low requirements for equipment and personnel skills, making it easy to promote and apply in grassroots seed management departments, seed companies, and breeding units, providing practical technical support for regulating the seed market and ensuring food production security. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Gel electrophoresis images of maize varieties identified using molecular markers 1-9, including 16 maize varieties such as Ludan 510, Zhengdan 958, and Ludan 517. Figure 2 Gel electrophoresis images of maize varieties identified using molecular marker 10-17, including 16 maize varieties such as Ludan 510, Zhengdan 958, and Ludan 517. Figure 3 Gel electrophoresis images of maize varieties identified using molecular marker 18-25 pairs, including Ludan 510, Zhengdan 958, and Ludan 517. Figure 4 Gel electrophoresis images of 16 maize varieties, including Ludan 510, Zhengdan 958 and Ludan 517, identified by molecular marker 26-33. Figure 5 Gel electrophoresis images of 16 maize varieties, including Ludan 510, Zhengdan 958 and Ludan 517, identified by molecular marker 34-41. Figure 6 Gel electrophoresis images of maize varieties identified using molecular markers 42-49, including 16 maize varieties such as Ludan 510, Zhengdan 958, and Ludan 517. Figure 7 Gel electrophoresis images of maize varieties identified using molecular markers 50-57, including Ludan 510, Zhengdan 958, and Ludan 517. Figure 8 Gel electrophoresis images of maize varieties identified using molecular markers 58-65, including 16 maize varieties such as Ludan 510, Zhengdan 958, and Ludan 517. Figure 9 Gel electrophoresis image of 16 maize varieties, including Ludan 510, Zhengdan 958 and Ludan 517, identified by molecular marker 66-73. Figure 10 Gel electrophoresis images of maize varieties identified using molecular markers 74-80, including 16 maize varieties such as Ludan 510, Zhengdan 958, and Ludan 517. Figures 1-10The electrophoresis bands in the middle, from left to right, are Maker, LuDan 510, LuDan 503, LuDan 506, LuDan 507, LuDan 509, LuDan 513, HuaYu 802, HuaYu 806, ZhengDan 958, LuDan 5211, LuDan 5212, LuDan 5223, LuDan 5225, LuDan 5226, LuDan 5232, and LuDan 5237. Figure 11 A phylogenetic tree was constructed based on the genotypes of 16 varieties at 80 InDel loci. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The maize materials used in the embodiments of this invention are all from the maize germplasm bank of Shandong Academy of Agricultural Sciences.

[0026] Example This embodiment details the screening process, primer design, experimental methods, and application of the InDel molecular marker combinations for maize variety identification described in this invention in variety identification and genetic relationship analysis.

[0027] 1. Screening and primer design of InDel markers To obtain a set of InDel marker combinations with high polymorphism and high discriminative power, this invention follows the screening process as follows: (1) Material selection and sequencing: A total of 150 maize accessions with broad genetic representativeness were collected, including backbone inbred lines (Qixi 5101, Qixi 5102, etc.) and main hybrids (Zhengdan 958, Xianyu 335, etc.). Genomic DNA was extracted from each material, and whole-genome resequencing was performed using the Illumina sequencing platform, with an average sequencing depth of 30×. The maize reference genome B73 RefGen_v5 was used as a reference.

[0028] (2) Site identification: The HaplotypeCaller module in the GATK (Genome Analysis Toolkit) software package was used to compare and detect variants in the resequencing data, and a total of 186,742 original InDel sites were identified.

[0029] (3) Screening criteria: To meet the requirements of high discriminative power, high stability, and convenient experimentation for variety identification, the above-mentioned loci were subjected to multiple rounds of rigorous screening. The specific criteria included: Insertion or deletion fragments must be ≥ 50 bp in length to ensure that different alleles can be clearly distinguished by ordinary agarose gel electrophoresis; Minor allele frequency (MAF) ≥ 0.3 ensures that the locus has a high amount of polymorphism information in the population; The loci are distributed as evenly as possible across the 10 chromosomes of maize to cover the entire genome and improve the overall effectiveness of the identification system. Flanking sequences (usually about 150-250 bp upstream and downstream of the mutation site) are highly unique, making it easy to design specific polymerase chain reaction (PCR) primers.

[0030] (4) Marker combination determination: Through the above screening, 80 InDel molecular markers that meet the criteria were finally obtained, which constituted the core marker combination. Detailed information about these markers, including their chromosomal location, location in the reference genome, and the fragment size (unit: base pairs, bp) of the two major alleles, is listed in Table 1.

[0031] Table 1. 80 InDel molecular markers that meet the criteria obtained through screening. (5) Primer design: Specific PCR amplification primers were designed for the conserved flanking sequence regions of the 80 InDel markers mentioned above. Primer Premier 5.0 software was used for design to ensure primer specificity and to ensure that each primer pair worked at similar annealing temperatures (Tm values). Table 2 shows the corresponding primer sequences for all 80 sites (sequence orientation 5'-3').

[0032] Table 2. Primer sequences for all 80 sites. 2. Using molecular markers for maize variety identification Using genomic DNA from 20 different maize varieties (including Ludan 510, Zhengdan 958, and Ludan 517) as templates, PCR amplification was performed using the aforementioned 80 primer pairs to verify the effectiveness of the markers and construct variety fingerprint profiles.

[0033] (1) PCR amplification: Reaction system: The total volume is 20 μL, and the specific composition is shown in Table 3. The 2× Taq PCR MasterMix contains DNA polymerase, dNTPs, and Mg... 2+ And necessary components such as buffer solution.

[0034] Reaction procedure: Run on a standard PCR instrument, and the program settings are shown in Table 4. The annealing temperature can be fine-tuned within the range of 58-62℃ based on the actual Tm value of the primers.

[0035] Table 3 PCR reaction system (20 μL) Table 4 PCR reaction procedure (2) Electrophoresis detection: Take 5 μL of PCR product and separate it by electrophoresis using a 2% agarose gel containing nucleic acid dye. After electrophoresis at a constant voltage of 120V for about 25 minutes, observe and photograph the product under a gel imaging system (or UV transilluminator).

[0036] (3) Genotype interpretation: The genotype of each sample at each InDel locus is determined based on the position of the electrophoretic bands (i.e., the size of the DNA fragment). Homozygotes show a single condition, and heterozygotes show two conditions. The band size should correspond to the allele size in Table 1.

[0037] Electrophoresis results as follows Figures 1-10 As shown.

[0038] 3. Fingerprint pattern construction and variety identification analysis (1) Fingerprinting: The detection results of 80 InDel markers for 16 tested varieties (LD510, LD503, etc.) were systematically recorded. The genotype of each variety at each locus (represented as "allele 1 size | allele 2 size") constituted the unique digital fingerprint of that variety. Data examples are shown in Table 5. This set of markers can effectively distinguish all tested varieties, and even closely related varieties (such as sister lines from the same population) show distinguishable genotypic differences.

[0039] Table 5. Statistical analysis of genotypes in 16 samples (2) Genetic Relationship Analysis: Genotypic data ("0 / 1" or similar format) of the above 16 varieties at 80 InDel loci were imported into bioinformatics analysis software (such as PowerMarker, NTSYS, or R language). Cluster analysis was performed using methods such as Unweighted Group Mean Method (UPGMA) to construct a phylogenetic tree. Analysis results (such as...) Figure 11 The results show that varieties with similar genetic backgrounds (such as derivatives with common parents) can be correctly clustered together, indicating that the InDel marker combination of the present invention can not only be used for the unique identification of variety identity, but also effectively reflect the genetic relationship between varieties, and can be used for germplasm resource analysis and breeding population division.

[0040] This invention provides a complete and operable InDel-based molecular marker-based maize variety identification technology. This method utilizes 80 highly polymorphic InDel markers obtained through screening to achieve rapid and accurate variety identification and fingerprint construction via conventional PCR and agarose gel electrophoresis. The entire method requires no complex instruments, is low-cost, and provides intuitive and reliable results. It is particularly suitable for promotion and application in grassroots seed management stations, small and medium-sized enterprises, and breeding units. It can serve as an effective supplement to current national standards (based on SSR markers), providing strong technical support for maize seed quality supervision, variety rights protection, and market regulation.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An InDel molecular marker combinatorial for maize germplasm / variety identification, characterized in that, The InDel molecular marker combination consists of InDel sites located at B73 of the reference genome, as shown in the table below: 。 2. A primer set for amplifying the InDel molecular marker combination of claim 1, characterized in that, It consists of primers with nucleotide sequences as shown in SEQ ID NO.1-160.

3. A kit comprising the primer set of claim 2.

4. The application of the InDel molecular marker combination of claim 1, the primer set of claim 2, or the kit of claim 3 in constructing fingerprint profiles of maize varieties.

5. A method for constructing a fingerprint profile of a maize variety, characterized in that, Includes the following steps: (1) Extract genomic DNA from maize variety samples to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in claim 2 or the kit described in claim 3; (3) Perform electrophoresis on the amplification products of step (2) and determine the genotype of each site of the maize variety in the InDel molecular marker combination described in the claims based on the position of the electrophoretic bands; (4) The genotype information obtained in step (3) is digitally recorded to form a specific fingerprint map of the maize variety to be tested.

6. The application of the InDel molecular marker combination of claim 1, the primer set of claim 2, or the kit of claim 3 in maize variety identification.

7. A method for identifying maize varieties, characterized in that, Includes the following steps: (1) Extract genomic DNA from the maize sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer set described in claim 2 or the kit described in claim 3; (3) Perform electrophoresis detection on the amplification products of step (2) and determine the genotype of each site in the InDel molecular marker combination described in the claims based on the position of the electrophoretic bands; (4) Compare the genotype obtained in step (3) with the fingerprint of known maize varieties, and determine the variety identity of the maize sample to be tested based on the comparison results.

8. The application of the InDel molecular marker combination of claim 1, the primer set of claim 2, or the kit of claim 3 in the detection of genetic kinship among maize varieties.

9. The application of the InDel molecular marker combination of claim 1, the primer set of claim 2, or the kit of claim 3 in the analysis of maize germplasm resources.

10. The application of the InDel molecular marker combination of claim 1, the primer set of claim 2, or the kit of claim 3 in maize breeding population segmentation or variety uniformity identification.

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