A maize genotyping primer combination suitable for nanopore sequencing platform and application thereof

Abstract

This invention discloses a primer combination for maize genotyping suitable for nanopore sequencing platforms and its application. The primer combination includes at least one of 11 pairs of ONT primers, the sequences of which are shown in SEQ ID NO: 1-SEQ ID NO: 22. The primers of this invention can specifically amplify target regions rich in genetic variation in the maize genome. The amplified PCR products are mixed and then sequenced using nanopore sequencing. By analyzing the species attributes and genome alignment positions of the sequencing sequences, combining experimental data from the test samples to calculate the targeting rate of the amplified products, and associating them with known genomic information, the purity and identification accuracy of maize seeds can be determined. This primer combination combines maize material specificity with population compatibility, enabling economical and efficient genotyping of maize inbred lines and hybrid populations. It can also be applied to verify the authenticity of maize varieties, analyze the genetic background of breeding materials, and screen for genotyping.

Description

Technical Field

[0001] This invention relates to the field of maize molecular detection and molecular breeding technology, and in particular to a maize genotyping primer combination and its application suitable for nanopore sequencing platforms. Background Technology

[0002] With the development of molecular biology, the molecular markers used for maize genotyping are also undergoing rapid evolution. From first-generation molecular markers such as RFLP (restriction fragment length polymorphism) and SSR (simple sequence repeats, or microsatellites), to those centered on SNP (single nucleotide polymorphism), genotyping has entered the high-throughput era. Gene chip technology and single SNP marker detection systems, such as TaqMan and KASP markers, are currently commonly used genotyping methods. However, KASP can only detect a single or a small number of pre-designed loci. When the number of loci to be detected increases, the cost and experimental complexity of both custom-designed chips and multiplex fluorescent PCR increase significantly, forming a throughput bottleneck.

[0003] Nanopore sequencing is a representative of third-generation single-molecule real-time sequencing technology. Its core principle involves guiding a single DNA or RNA molecule through nanoscale protein pores embedded in a polymer membrane via electrophoresis. As different bases pass through, they characteristically interfere with the ionic current within the pores. By monitoring and decoding these current changes in real time, the nucleic acid sequence can be directly determined. The most prominent advantages of this technology are its ultra-long read lengths (current platforms easily generate continuous reads of tens of thousands to millions of bases) and real-time performance (data output is almost synchronous with the sequencing process). However, due to current sequencing cost limitations, nanopore sequencing technology has not yet been used in crop genotyping. Summary of the Invention

[0004] To address the problems existing in current maize genotyping technologies, this invention provides a maize genotyping primer combination and its application suitable for nanopore sequencing platforms.

[0005] Specifically, the technical solution adopted in this invention is as follows:

[0006] This invention is based on a proprietary primer bioinformatics design and analysis workflow. Using publicly available third-generation sequencing genome sequences from the MaizeGDB database as a foundation, and considering the genetic diversity characteristics among different maize inbred lines, it screens target regions through bioinformatics analysis, selects conserved nucleotide sequences from multiple genomes as amplification regions, and designs specific primers suitable for nanopore sequencing. These primers include at least one of 11 pairs of ONT primers, the sequences of which are shown in SEQ ID NO: 1-SEQ ID NO: 22.

[0007] This primer can specifically amplify target regions with rich genetic variation in the maize genome. The amplified PCR products are mixed and then sequenced in nanopores. By analyzing the species attributes and genome alignment positions of the sequencing sequences, combined with the experimental data of the test samples, the targeting rate of the amplified products is calculated, and known genomic information is associated, so as to determine the purity and identification accuracy of maize seeds.

[0008] Experiments on the specificity and sensitivity of the primers confirmed that the primer combination of the present invention can be used for rapid, convenient and efficient genotyping. After equal amounts of amplification products are mixed and analyzed by nanopore sequencing, maize can be accurately and efficiently genotyped.

[0009] Compared with the prior art, the outstanding effect of the present invention is as follows:

[0010] This invention employs a genotyping strategy combining conventional PCR amplification and nanopore sequencing. By designing specific primers to amplify target regions rich in genetic variation within the maize genome, multiple amplification products are mixed and then used for a single library construction and nanopore sequencing run. This strategy achieves a very high degree of multiplexing, significantly reducing the detection cost per site. Simultaneously, leveraging the long read length of nanopore sequencing, new genetic variations can be discovered while performing genotyping, overcoming the limitations of existing technologies in terms of discovery capabilities.

[0011] Furthermore, nanopore sequencing's advantages of "rapid library construction and real-time data output" can shorten the entire genotyping cycle to within hours, significantly improving detection efficiency. The method of this invention enables more accurate, efficient, and low-cost acquisition of maize genetic information, providing strong support for maize genetic structure analysis and precision breeding practices.

[0012] The primer combination of this invention combines maize material specificity with intrapopulation compatibility, enabling economical and efficient genotyping of maize inbred lines and hybrid populations. It can also be applied to verify the authenticity of maize varieties, analyze and screen the genetic background of breeding materials, and has broad prospects for industrial application.

[0013] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the maize genotyping primer combinations suitable for nanopore sequencing platforms and their applications according to the present invention. Attached Figure Description

[0014] Figure 1 Electrophoresis results of PCR amplification products of 16 ONT primers are shown. Leaf DNA was used to detect the specificity and amplification efficiency of the 16 ONT primer pairs. Sample numbers 1, 2, 3, 4, 5, and 6 correspond to the material names PH6WC, WK858, WK798-2, PH4CV, Jing724, and Jing92, respectively.

[0015] Figure 2 Electrophoresis results of PCR amplification products from 34 maize inbred line DNA samples using five ONT primers (ONT01, ONT02, ONT09, ONT10, and ONT11) are shown in Table 3 (DNA sequence is shown). These results support the sequence number obtained by nanopore sequencing.

[0016] Figure 3 This is a flowchart of the method in Embodiment 3 of the present invention. Detailed Implementation

[0017] Example 1: Design and evaluation of primer sequences and target amplification regions

[0018] Based on the 50 maize third-generation sequencing genomes published by MaizeGDB (see Table 1 for details), sequence alignment analysis was used to exclude secondary structure and non-specific sequence regions, selecting the most genetically varied specific sequence segments. Conserved nucleotide sequences were chosen as amplification regions, while ensuring the richest genetic variation in the amplified fragments, thus enabling variety-specific differentiation of the targeted amplification regions. Furthermore, given that the sequencing technology used in this application is nanopore sequencing, which involves long-fragment amplification and has strict requirements on the minimum amplified fragment size, all primer pairs in this application can amplify fragments of over 500 bp. After optimizing the amplification efficiency, 16 ONT primer markers were developed using mIndel software (Lv Y, Liu Y, Zhao H. mInDel: a high-throughput and efficient pipeline for genome-wide InDel marker development. BMC Genomics. 2016. 14;17:290.) and existing primer bioinformatics design and analysis workflows (primer sequences are shown in SEQ ID NO: 1-SEQ ID NO: 32), as detailed in Table 2. Further evaluation of the binding ability of the designed primers to the template was conducted. Six high-quality maize inbred lines (PH6WC, WK858, WK798-2, PH4CV, Jing724, and Jing92) were randomly selected to test the 16 ONT primers to be tested. A total of 11 pairs of ONT primers with single bands and high amplification efficiency were identified. Figure 1 The primer sequences are shown in SEQ ID NO: 1-SEQ ID NO: 22.

[0019] Table 1. Information on 50 maize third-generation sequencing genome materials published by MaizeGDB used in this invention.

[0020]

[0021] Table 2 Information on 16 ONT primer markers

[0022] ;

[0023] Note: Primer location information and PCR amplification product length are based on B73 (V5) as the reference gene.

[0024] Example 2: Genotyping of maize inbred line samples

[0025] DNA was extracted from leaves of 34 maize inbred lines with publicly available genomes from MaizeGDB using the TPS method. Eleven pairs of ONT primers were added to the PCR amplification reaction system, followed by amplification. The PCR amplification system (20 µL) consisted of: 12.5 µL of 2 × PCR Mix, 1.0 µL each of forward and reverse primers (10 µmol / L), 1 µL of genomic DNA, and 4.5 µL of ultrapure water. The PCR program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 5 min. After amplification, 10 µL portions of the PCR products from the 11 pairs of ONT primers from the same sample were mixed. Beijing Qingke Biotechnology Co., Ltd. constructed libraries from the 96 mixed amplification products and further sequenced them using Ultra Sequencing technology at a sequencing depth of 200X.

[0026] The obtained nanopore long read data were first quality controlled, filtering out low-quality and excessively short sequences, and removing adapter sequences. The number of nanopore sequences (N) containing ONT primer sequences obtained for each sample was counted. After sequence quality control, BLAST was used to align the genomes of 34 maize inbred lines T2T published in MaizeGDB, outputting results in outfmt6 format. Then, the BLAST output results were optimized based on parameters such as identity (%) > 98%, e-value > 10e-40, and bit score (highest) to obtain the alignment position of the sequences on the reference genome, and to determine the optimal reference genome alignment and the number of aligned sequences (Target-N). Based on the statistical results obtained in the previous step, the Target-R / R ratio was calculated using the number of sequences amplified by the 11 ONT primers (N) and the number of sequences aligned to the target region (Target-N) to obtain the targeting rate.

[0027] The detection results of the samples are shown in Table 3. It can be seen that the number of sequencing sequences obtained by PCR amplification using 11 pairs of ONT primers is 100% consistent with the results aligned to the target reference genome. Therefore, the primers designed in this invention can efficiently and specifically perform maize genotyping with 100% specificity.

[0028] Table 3. Validation of nanopore typing results for maize inbred line samples from 34 public databases.

[0029] ;

[0030] Example 3: Method for detecting maize genotypes using 11 ONT primers

[0031] This invention amplifies specific regions of maize rich in genetic variation by designing nanopore primers. The resulting PCR products are then mixed and sequenced using nanopore sequencing. By analyzing the species attributes and alignment positions of the sequences, the purity and accuracy of the seeds are determined based on the experimental results of the test samples and the target rate of the amplified products and known genomic information.

[0032] like Figure 3 As shown, the specific steps are as follows:

[0033] 1. Take R5 stage leaves from 102 maize samples to be tested, and extract plant genomic DNA using the TPS method. Grind the samples, precipitate, wash and dissolve to finally obtain genomic DNA.

[0034] 2. Construct amplification reaction systems by adding ONT primers separately, followed by PCR amplification. PCR amplification system (50 µL): 25 µL 2 × PCR Mix, 1.0 µL each of forward and reverse primers (10 µmol / L), 1 µL genomic DNA, and 22 µL ultrapure water. The PCR program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles, with a final extension at 72℃ for 5 min.

[0035] 3. After amplification, 10 µL of the PCR product from the 11 pairs of ONT primers of the same sample is taken and mixed.

[0036] Fourth, Beijing Qingke Biotechnology Co., Ltd. constructed libraries for 102 mixed amplification products and further sequenced them using Ultra (nanopore sequencing) technology.

[0037] 5. The obtained nanopore long read data are first subjected to quality control to filter out low-quality and excessively short sequences, while removing adapter sequences, and the number (N) of nanopore sequencing sequences containing ONT primer sequences obtained for each sample to be tested is counted.

[0038] VI. After sequence quality control, the data were compared with the T2T genome data of our team's 102 maize inbred line using BLAST, and the results were output in outfmt 6 format.

[0039] 7. Based on parameters such as Identity (%) > 98%, E-value > 10e-40, and Bit score (maximum value), the blastn output results are selected to obtain the alignment position of the sequence on the reference genome, and to determine the optimal reference genome and the number of aligned sequences (Target-N).

[0040] 8. Based on the above statistical results, the ratio of Target-R / R is calculated by further using the number of sequences amplified by the 11 ONT primers (N) and the number of sequences aligned to the target region (Target-N) to obtain the targeting rate.

[0041] The detection results of the samples are shown in Table 4. It can be seen that the number of sequencing sequences obtained by PCR amplification of 11 pairs of ONT primers is consistent with the known genomic information in 99.01% of the tested samples (101 inbred lines) compared with the results aligned to the target reference genome. This shows that the genotyping method based on nanopore sequencing of this invention can efficiently and specifically identify the accuracy and purity of maize materials.

[0042] Table 4. Nanopore detection results of 102 maize inbred line samples

[0043] ;

[0044] ;

[0045] ;

[0046] 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. The application of a maize genotyping primer combination suitable for nanopore sequencing platforms in the construction of maize variety DNA fingerprint databases, identification of maize variety authenticity and seed purity detection, cluster analysis of maize germplasm resources, genetic background analysis and screening of new maize breeding materials, and marker-assisted breeding of maize, characterized in that: It includes 11 pairs of ONT primers, the sequences of which are shown in SEQ ID NO: 1-SEQ ID NO:

22.

2. The application according to claim 1, characterized in that: The primer combination described in claim 1 is used to specifically amplify the target region with rich genetic variation in the maize genome. The amplified PCR products are mixed and then sequenced in a nanopore. By analyzing the species attributes and genome alignment positions of the sequencing sequences, the target rate of the amplified products is calculated by combining the experimental data of the test samples, and the known genomic information is associated, so as to determine the purity and identification accuracy of maize seeds.

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