InDel marker set capable of being used for identifying authenticity of corn variety

Abstract

The invention relates to the technical field of crop molecular biology, in particular to an InDel marker set capable of being used for identifying the authenticity of corn varieties. According to the invention, a set of InDel marker set compatible with multiple platforms and used for identifying the authenticity of corn varieties is developed from the whole genome level, and primers suitable for capillary electrophoresis and KASP platforms are designed. According to the method, the range of available marker sites of corn is expanded on the genome level, and meanwhile, a new thought and a new marking method are provided for researches such as construction of a corn DNA fingerprint database, identification of corn variety authenticity and the like.

Description

Technical Field

[0001] This invention relates to the field of crop molecular biology technology, and in particular to a set of InDel markers that can be used for the identification of maize varieties. Background Technology

[0002] The rapid development of high-throughput sequencing, bioinformatics, and genotyping technologies has made it possible to effectively identify crop varieties using newer and more efficient molecular techniques. Traditional molecular markers, especially SSR markers, have been the mainstream markers for crop molecular identification in the past due to their high polymorphism, numerous alleles, and ease of detection. However, the low distribution density of SSR markers and the difficulty in data sharing hinder large-scale and automated implementation, limiting their large-scale application in crop molecular detection. InDel, an insertion / deletion polymorphism marker, is widely distributed and densely distributed in the genome; in terms of distribution density, InDel is second only to SNP, but far higher than SSR. Compared with SNP, both belong to biseleural polymorphism, and 90% of InDel are small InDels (InDel sequence length less than 10 bp). As a new generation of genetic markers, it has length differences and biseleural variation characteristics, and is compatible with multiple genotyping detection platforms. Specifically: First, based on length difference characteristics, the capillary electrophoresis technology platform, widely used in maize molecular detection laboratories, can be applied. This genotyping technique is easy to master and popularize; furthermore, the alleles are fixed and known, allowing detection using very small amplicons, thus facilitating the construction of multiplex PCR amplification and electrophoresis detection. Second, for insertion / deletion biseleural variants, the high-throughput KASP platform can be used for genotyping. Third, high-throughput microarray platforms for both loci and samples can also be used for genotyping; genotyping data from all three platforms can be easily integrated across platforms.

[0003] Given that different varieties have different molecular structures requiring different genotyping platforms, there is an urgent need to develop a multi-platform compatible InDel marker set for maize variety authenticity identification. In existing technologies, CN112280884A and CN110846429A both disclose InDel marker sets for maize genotyping, but the number of InDel markers involved is over 1000. In practical applications, this results in a large number of markers and redundancy; genotyping can only be performed using microarray or sequencing platforms, and cannot utilize simpler and faster capillary electrophoresis or KASP platforms. Summary of the Invention

[0004] This invention collects 300 maize hybrid materials from a wide range of sources, with rich phenotypes and genotypes and high representativeness. InDel genome chip analysis is performed on these materials. Based on the experimental quality and using the optimal genetic algorithm, a set of 96 InDel markers suitable for maize variety authenticity identification is selected. For the developed loci, primers suitable for both capillary electrophoresis and KASP platforms are designed and evaluated, providing technical support for the construction of a maize variety fingerprint database and authenticity identification. Based on this, the following technical solution is proposed.

[0005] In one aspect, the present invention provides a set of InDel markers for identifying the authenticity of maize varieties, as shown in Table 1. The information in square brackets indicates deleted or inserted base sequences. The reference genome version number for chromosomes and physical locations in Table 1 is B73 RefGen_v3.

[0006] Table 1 The determination of the aforementioned InDel marker set is the key data and result of this invention. The accuracy and efficiency of obtaining InDel loci are ensured through representative sample selection, high-quality microarray data, and precise data analysis. This invention selects 300 materials from a wide range of sources with rich phenotypes and genotypes. Based on whole-genome microarray data and utilizing the optimal genetic algorithm, the aforementioned InDel polymorphic locus set was finally determined. With only 96 InDel polymorphic loci, accurate identification of maize variety authenticity can be achieved. More importantly, this InDel polymorphic locus set is applicable to both the simple and rapid capillary electrophoresis and KASP platforms.

[0007] Secondly, the present invention provides a primer set for detecting the InDel marker set.

[0008] Preferably, the primer sets are shown in Table 2 or Table 3. The primer sets shown in Table 2 are suitable for capillary electrophoresis platforms; the primer sets shown in Table 3 are suitable for KASP platforms.

[0009] Table 2 Table 3 The detection technology established using the aforementioned high-throughput dual-platform primer set provides a good method for constructing the InDel-DNA fingerprint database and also provides database support for the authenticity identification of maize varieties.

[0010] Thirdly, the present invention provides a molecular probe for detecting the InDel-tagged set.

[0011] Fourthly, the present invention provides a gene chip containing the molecular probe.

[0012] Preferably, the gene chip is a solid-phase chip or a liquid-phase chip.

[0013] Fifthly, the present invention provides a kit containing the primer set, or the molecular probe, or the gene chip.

[0014] In a sixth aspect, the present invention provides the application of the InDel marker set, the primer set, the molecular probe, the gene chip, or the kit in constructing a maize variety InDel-DNA fingerprint database.

[0015] In a seventh aspect, the present invention provides the application of the InDel marker set, the primer set, the molecular probe, the gene chip, or the kit in the identification of maize variety authenticity.

[0016] Eighthly, the present invention provides a method for constructing a maize InDel-DNA fingerprint database, comprising: (1) Extract genomic DNA from each maize variety to construct the fingerprint database; (2) Using the primer set for detecting the InDel marker set, the InDel-DNA fingerprint data of each maize variety is obtained based on the fluorescent capillary electrophoresis platform or the KASP platform, thereby constructing a maize InDel-DNA fingerprint database.

[0017] Ninthly, the present invention provides a method for identifying the authenticity of maize varieties, comprising: performing PCR amplification using a primer set for detecting the InDel marker set to construct a fingerprint database of different maize varieties; wherein the fingerprint database contains fingerprint data of the maize variety to be identified; The genome of the maize variety to be identified was amplified by PCR using the primer set for detecting the InDel marker set to obtain fingerprint data of the maize variety to be identified. The fingerprint data of the corn variety to be identified is compared with the fingerprint database to determine the corn variety.

[0018] Preferably, PCR amplification is performed using primers from a capillary electrophoresis platform or a KASP platform, and genotype data is collected from the amplified products to construct a fingerprint database for different maize varieties.

[0019] In the specific implementation process, the primer sets in Tables 2 and 3 can be used to obtain genotyping data on a fluorescent capillary electrophoresis or KASP platform. The specific procedure is as follows: label the 5' end of one primer in each primer pair with a fluorescent group or add a fluorescent adapter sequence to the two forward primers in the KASP pair; prepare the PCR reaction system by mixing DNA, primers, dNTPs, MgCl2, Taq enzyme, and buffer; run the reaction program; detect the amplification products using a fluorescent capillary electrophoresis system or a fluorescence scanner; collect the raw data using the capillary electrophoresis system or the software accompanying the KASP platform, and import the raw data into genotyping software to obtain genotyping data.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: Based on the characteristics of InDel markers, this invention develops a set of InDel markers compatible with multiple platforms at the whole-genome level for maize variety authenticity identification, and designs primers suitable for both capillary electrophoresis and KASP platforms. This invention expands the range of available marker sites in maize at the genome level, and provides new ideas and marker methods for the construction of maize DNA fingerprint databases and maize variety authenticity identification. Attached Figure Description

[0021] Figure 1 This is an experimental result based on capillary electrophoresis and the KASP platform InDel labeling (MIDEK27); the left side is the result of the capillary electrophoresis platform, with the amplified fragment length being 230 / 233, corresponding to allelic variations of D / I; the right side is the result of the KASP platform, with red representing DD, blue representing II, and green representing heterozygous DI. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0023] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals accompanying various commercial reagent kits or entrust them to specialized companies, such as gene sequencing, microarray preparation, and data analysis.

[0024] Example 1: Determination of the InDel marker set for maize variety authenticity identification This embodiment provides an InDel marker set that can be used for the identification of maize varieties, and develops primers for capillary electrophoresis and the KASP platform. The steps are as follows: (1) Select 300 representative maize experimental materials, including the nationally approved hybrid varieties currently being promoted in my country. (2) Prepare high-concentration, high-quality total DNA. (3) Evaluate the site test results based on the detection data of the whole genome InDel chip. (4) Obtain a set of 96 InDel sites using the optimal genetic algorithm. (5) Primer design: Use Primer 5 software to design primers based on capillary electrophoresis and the KASP platform. (6) Primer evaluation and verification: Select 30 representative samples of various types to evaluate and verify the primers based on capillary electrophoresis and the KASP platform. (7) Based on the experimental data of the above 30 samples, evaluate the following aspects: whether the primer amplification was successful, whether it was consistent with the high-density chip results, and whether it had polymorphism. The evaluation results are as follows: Figure 1 As shown, the results indicate that 96 pairs of electrophoresis primers exhibited good amplification performance on the capillary electrophoresis platform, and 96 pairs of KASP primers exhibited good amplification performance on the KASP platform. Information on the InDel site and dual-platform primers developed in this invention is shown in Table 4 below.

[0025] Table 4 Note 1: For capillary electrophoresis platforms, small allele fragments are recorded as D and large allele fragments are recorded as I when recording genotypes.

[0026] Note 2: In KASP primers, primer sequences labeled with FAM fluorescence (blue) represent insertion allelic variations and are recorded as I; primer sequences labeled with HEX fluorescence (green) represent deletion allelic variations and are recorded as D. Homozygous genotype II is shown as a blue fluorescent signal, homozygous genotype DD is shown as a green fluorescent signal, and heterozygous genotype DI is shown as both blue and green fluorescent signals.

[0027] Example 2 uses the InDel marker set from Example 1 to construct an InDel-DNA fingerprint database for maize varieties. (1) DNA extraction and quality identification Each sample was grown from seed, exposed to light, and allowed to develop into green seedlings. DNA extraction was performed using a mixed-plant method, combining green leaves from 30 individual plants. Total genomic DNA was extracted from the samples using the standard CTAB method. The DNA was diluted to prepare working solutions at concentrations of 200 ng / μL and 20 ng / μL.

[0028] (2) Experimental procedure of capillary electrophoresis platform Primer synthesis: Primers were synthesized based on the 96 successfully validated primer pairs provided in Table 4; one primer in each pair was labeled with a fluorescent group at its 5' end.

[0029] PCR amplification: The PCR reaction system consisted of 20 μL, including 2 µL of DNA working solution, 10 µL of 2×Taq Plus MasterMix, and 0.25 µL of primers. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 60℃ annealing for 35 s, and 72℃ extension for 45 s, for a total of 35 cycles; 72℃ extension for 10 min; and storage at 4℃.

[0030] Electrophoresis and Data Acquisition: After PCR amplification, 1 µL of the amplified product was directly added to the wells of a 96-well plate specifically designed for the DNA analyzer. 0.1 µL of molecular weight internal standard and 8.9 µL of deionized formamide were added to each well. The plate was denatured at 95°C for 5 min on a PCR instrument, then immediately placed on ice and cooled for at least 10 min. After brief centrifugation for 10 s, the plate was ready for use. Capillary electrophoresis was performed on an ABI 3730XL DNA analyzer. Raw data were collected using Data Collection V1.0 software. The raw data after electrophoresis were collected using the Data Collection V1.0 software included with the ABI 3730XL DNA analyzer and generated into an FSA file. Genotyping analysis of the FSA file was performed using the SSR Analyser V1.2.4 fingerprint analyzer.

[0031] (3) Experimental procedure of KASP platform Primer synthesis: Based on the 96 successfully validated primer pairs provided in Table 4, primers were synthesized; adapter sequences for FAM or HEX fluorescent groups were added to the 5' ends of the two forward primer sequences in each primer pair.

[0032] The PCR amplification reaction was performed using a 1536 microplate. The reaction volume was 1 µL, which contained 1.5 µL of DNA working solution (dried), 0.5 µL of 2 × Master mix, 0.486 µL of deionized water, and 0.014 µL of primer working solution.

[0033] PCR amplification reactions were performed on a water bath PCR instrument (Hydrocycler-64, LGC Genomics). The amplification reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, annealing at 61~55℃ for 60 s, 10 cycles (decreasing by 0.6℃ per cycle); 94℃ denaturation for 20 s, annealing at 55℃ for 60 s, 26 cycles. Each plate contained 96 samples, including 2 control samples, 1 replicate sample, and 1 blank control.

[0034] The amplified products were scanned using a fluorescence scanner (PHERAstar plate reader, LGC Genomics), and the genotype data were collected using Kraken software (V17.12.8.19596, LGC Genomics).

[0035] (4) Data comparison and entry into the database The genotyping results from the two platforms are compared. If they match, the data is directly entered into the database. If they do not match, a retest is conducted and the results are reassessed. Finally, the InDel-DNA fingerprint database of maize varieties is constructed.

[0036] Example 3: Using the InDel marker set from Example 1 for maize variety authenticity identification For the maize hybrid A sample (Jingke 968) to be identified, DNA was extracted from the green leaves of seedlings. PCR amplification was performed using primers from the 96 successfully verified InDel marker sets in Table 4. Fingerprint data was obtained by fluorescent capillary electrophoresis or KASP fluorescence scanning, using the same method as in Example 2.

[0037] Based on the comparison between the InDel fingerprint data of sample A and the standard fingerprint in the InDel marker fingerprint database of known maize varieties (constructed in Example 2, which contains the standard fingerprint of Jingke 968), it was determined that the InDel fingerprint data of the sample to be tested was the same as that of Jingke 968, indicating that hybrid sample A is Jingke 968 or a similar variety (Table 5). Furthermore, the above method was used to identify the authenticity of a large number of other unknown maize samples, and the results were consistent with the identification results of industry standard methods, demonstrating that the 96 InDel marker sets of this invention can quickly and accurately identify the authenticity of maize varieties.

[0038] Table 5

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A set of InDel tags, characterized in that, The InDel tag set is shown in Table 1 of the specification.

2. A primer set for detecting the InDel tag set of claim 1.

3. The primer set according to claim 2, characterized in that, The primer set is shown in Table 2 or Table 3 of the specification.

4. A molecular probe for detecting the InDel-tagged set of claim 1.

5. A gene chip containing the molecular probe of claim 4.

6. A reagent kit, characterized in that, The kit contains the primer set as described in claim 2 or 3, or the molecular probe as described in claim 4, or the gene chip as described in claim 5.

7. The application of the InDel marker set of claim 1, the primer set of claim 2 or 3, the molecular probe of claim 4, the gene chip of claim 5, or the kit of claim 6 in constructing a maize variety InDel-DNA fingerprint database.

8. The application of the InDel marker set of claim 1, the primer set of claim 2 or 3, the molecular probe of claim 4, the gene chip of claim 5, or the kit of claim 6 in the identification of maize variety authenticity.

9. A method for constructing a maize InDel-DNA fingerprint database, characterized in that, include: (1) Extract genomic DNA from each maize variety to construct the fingerprint database; (2) Using the primer set for detecting the InDel marker set described in claim 1, the InDel-DNA fingerprint data of each maize variety is obtained based on the fluorescent capillary electrophoresis platform or the KASP platform, thereby constructing a maize InDel-DNA fingerprint database.

10. A method for identifying the authenticity of a maize variety, characterized in that, include: PCR amplification was performed using the primer set for detecting the InDel marker set described in claim 1 to construct a fingerprint database for different maize varieties; The fingerprint database contains fingerprint data of maize varieties to be identified; the genome of the maize variety to be identified is amplified by PCR using the primer set for detecting the InDel marker set described in claim 1 to obtain fingerprint data of the maize variety to be identified. The fingerprint data of the corn variety to be identified is compared with the fingerprint database to determine the corn variety; Preferably, PCR amplification is performed using primers from a capillary electrophoresis platform or a KASP platform, and genotype data is collected from the amplified products to construct a fingerprint database for different maize varieties.

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