Method for tracing maize hybrid parentage and application thereof

Abstract

The present application relates to the cross field of crop molecular breeding and bioinformatics technology, and in particular to a method for tracing the parent of a corn hybrid and application thereof. The method provided by the present application screens and sorts the whole genome SNP site data of a corn hybrid, encodes the virtual characteristic sequence, uses the homologous alignment method to align the virtual characteristic sequence with the local database, and determines the parent of the hybrid according to the matching result. The method provided by the present application solves the industry problem that the traditional technology cannot directly use the public database to accurately identify the parent of the hybrid through the unique data conversion mechanism and algorithm. Compared with the method for identifying and tracing the parent by using the similarity, the accuracy of the result obtained by the present method is greatly improved. Meanwhile, the present method also provides an efficient solution for the authenticity identification of the variety, the protection of the germplasm resource and the breeding design.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of crop molecular breeding and bioinformatics, specifically providing a method for tracing the parentage of maize hybrids and its application. Background Technology

[0002] Common maize varieties are produced by crossing two inbred lines, and hybrid vigor can significantly improve yield, disease resistance, and environmental adaptability. Hybrid breeding requires years of screening from tens of thousands of inbred lines to select superior parents. However, frequent instances of counterfeiting and theft in the seed market seriously infringe upon the rights of breeders and disrupt market order. With advancements in breeding technology and the increasing market demand for high-quality seeds, the authentication of hybrid varieties has become a core requirement for ensuring seed quality, preventing the misappropriation of germplasm resources, and curbing the circulation of counterfeit seeds.

[0003] Current technologies mostly use molecular markers such as SNP and INDEL, as well as genome sequencing, to trace the origin of maize samples. However, current technologies for identifying maize hybrid parents have three limitations: First, molecular marker methods such as SSR and AFLP have low throughput and are difficult to distinguish closely related materials; second, whole genome sequencing is expensive and Blast whole genome sequencing is inefficient; and third, solid-phase microarray analysis relies on private reference databases and cannot utilize public sequence resources such as NCBI.

[0004] To address the aforementioned technical bottlenecks, this invention innovatively proposes a pathway to transform discrete genotype data of SNP sites on solid-phase chips into continuous virtual allelic sequences, breaking through the constraints of traditional analytical frameworks. Summary of the Invention

[0005] The purpose of this invention is to provide a method for tracing the parentage of maize hybrids and its application. The core of this invention lies in establishing a technical chain of "discrete genotype → continuous characteristic sequence → efficient BLAST matching".

[0006] This invention provides a method for tracing the parentage of maize hybrids, comprising the following steps:

[0007] 1) Obtain whole-genome SNP loci and corresponding genotype data from maize hybrids, screen the whole-genome SNP loci and corresponding genotype data, and then sort the whole-genome SNP loci and corresponding genotype data according to chromosome number and physical location.

[0008] Specifically, the genotype data obtained from the maize solid-phase chip Illumina Maize SNP50 were preprocessed: high-quality SNP loci were screened (locusts with a deletion rate >10% and minor allele frequency <0.05 were removed), and the screened SNP loci were strictly sorted according to chromosome number and physical location.

[0009] 2) After screening and sorting, the genotype data corresponding to the whole genome SNP loci are encoded into two characters, and the two characters are concatenated to obtain a virtual feature sequence;

[0010] Specifically, the genotype data encoding involves encoding the homozygous genotype AA as "AA", TT as "TT", and other homozygous genotypes similarly; the heterozygous genotype AT is innovatively split into two characters "A" + "T", and other heterozygous genotypes are similarly processed; missing data is filled with "N". By connecting the encoded two characters, a virtual feature sequence of double length is generated, which essentially represents a sample-specific genotype pattern map.

[0011] 3) The local database is constructed by comparing the virtual feature sequences using the homology comparison method. Based on the homology comparison results, the parent of the maize hybrid is determined, thereby realizing the tracing of the parent of the maize hybrid.

[0012] The local database constructed in step 3) includes virtual characteristic sequences of maize inbred lines. Specifically, the maize inbred lines include all existing maize inbred lines on the market.

[0013] Specifically, when using the homology alignment method (BLAST alignment), a task blastn-short mode was adopted, and the key parameter word_size=7 was set to accommodate the short sequence matching requirements. At the same time, thresholds for e-value, similarity, and sequence coverage were limited to meet different needs. Finally, the parent lines of the hybrid were determined based on the matching results, and genetically similar materials (such as sister lines) were identified through error matching analysis, providing a reference for breeding design.

[0014] This invention provides an application of the above-mentioned method for tracing the parentage of maize hybrids, the application including any one of the following:

[0015] 1) Parental origin analysis of maize hybrids;

[0016] 2) Marker-assisted breeding of maize;

[0017] 3) Verify the authenticity of the corn variety;

[0018] 4) Protection of maize germplasm resources;

[0019] 5) Identify the genotype of maize hybrid offspring.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The method provided by this invention transforms discrete maize solid-phase gene chip locus data into continuous allelic sequences that can be compared for homology through an original data conversion mechanism and algorithm. This solves the industry problem that traditional technologies cannot directly use public databases for accurate identification of hybrid parents. Compared with methods that use similarity identification and parental tracing, the accuracy of the results obtained by this method is greatly improved. At the same time, it also provides an efficient solution for variety authenticity identification, germplasm resource protection, and breeding design. Attached Figure Description

[0022] Figure 1 The comparison results of hybrids GB001-GB003 of this invention among 92 inbred lines are shown below; A is the comparison result of hybrid GB001, B is the comparison result of hybrid GB002, and C is the comparison result of hybrid GB003.

[0023] Figure 2 The diagram shows the heterozygosity distribution of the inbred lines of this invention and the heterozygosity analysis results of some samples from 854 inbred line samples; A is the heterozygosity distribution of the inbred lines, and B is the heterozygosity analysis results of some samples from 854 inbred lines.

[0024] Figure 3 The comparison results of hybrids GB012-GB014 of the present invention among 854 inbred lines are shown in Figure 1; A is the comparison result of hybrid GB0012, B is the comparison result of hybrid GB0013, and C is the comparison result of hybrid GB0014. Detailed Implementation

[0025] The following specific embodiments further illustrate the technical solution of the method for tracing the parentage of maize hybrids and its application.

[0026] Example 1

[0027] (1) In the spring of 2023, in Xinxiang, Henan Province, a maize-producing area in the Huang-Huai-Hai Plain, 92 inbred lines and 31 hybrids used for crossbreeding were planted. Each inbred line and hybrid was planted in one row, with a row length of 5 meters and a total of 21 plants. Before maize pollination, leaves from each row were collected and sent to Wuhan Shuanglvyuan Company for DNA extraction and maize 50K chip detection. Genotypic data of 37,194 SNP loci in the whole genome of each of the 123 materials were obtained. Subsequently, SNP loci with poor quality (deletion rate >10% and minor allele frequency <0.05) were removed, and finally genotypic data of 35,623 SNP loci in the whole genome of each material were obtained. The 31 hybrids and their corresponding parents are shown in Table 1, which were used to verify the accuracy of the results.

[0028] Table 1. Hybrid Numbers and Their Parents

[0029] Test number - hybrid Test number - Parent 1 Test number - Parent 2 GB001 GB067 GB086 GB002 GB067 GB087 GB003 GB087 GB086 GB004 GB087 GB084 GB005 GB083 GB079 GB006 GB085 GB072 GB007 GB077 GB071 GB008 GB080 GB075 GB009 GB080 GB068 GB010 GB095 GB097 GB011 GB097 GB096 GB012 GB094 GB092 GB013 GB093 GB088 GB014 GB091 GB095 GB015 GB092 GB089 GB016 GB101 GB102 GB017 GB109 GB101 GB018 GB102 GB109 GB019 GB110 GB106 GB020 GB081 GB082 GB021 GB099 GB107 GB022 GB098 GB100 GB023 GB108 GB106 GB024 GB042 GB032 GB025 GB037 GB032 GB026 GB032 GB039 GB027 GB042 GB039 GB028 GB041 GB038 GB029 GB034 GB033 GB030 GB036 GB035 GB031 GB040 GB037

[0030] (2) Due to the high heterozygosity of hybrids, direct similarity analysis with inbred lines yields unsatisfactory results. In this embodiment, the genotype information of SNP loci is encoded and converted into two-character representations. Specifically, the homozygous genotype AA is encoded as "AA", TT is encoded as "TT", and other homozygous genotypes are represented similarly; the heterozygous genotype AT is innovatively split into two-character representations "A" + "T", and other heterozygous genotypes are represented similarly; missing data is filled with "N". A virtual feature sequence of double length is generated by connecting the encoded two-character representations. In this embodiment, the 35,623 SNP loci in each of the 123 materials are encoded into two-character representations, doubling the data to 71,246 data points per material.

[0031] (3) The 71246 data points of each material in the 92 inbred lines were concatenated and presented in FASTA format. Each inbred line material formed a virtual feature sequence. A local database was built using the BLAST software with the code `makeblastdb -dbtype nucl-parse_seqids -in 92.fasta -out 92inbred`. The virtual feature sequences of each material in the 31 hybrids were formed using the same method. Then, the virtual feature sequences of the 31 hybrids were compared in the local database with the code `blastn -query F1.fasta -db 92inbred -out result.txt -outfmt7 -max_hsps 1`.

[0032] (4) The comparison results of the hybrids GB001-GB003 of this invention in 92 inbred lines are as follows: Figure 1 As shown in (AC), Figure 1 The A-level results show that the highest scores for hybrid GB001 were achieved by inbred lines GB067 and GB086. Figure 1 B shows that the highest comparison scores for hybrid GB002 are for inbred lines GB067 and GB087. Figure 1 The C-value shows that the highest comparison score for hybrid GB003 is achieved by inbred lines GB086 and GB087, which are the male / female parents of these hybrids. This verifies the high accuracy of the method provided by this invention in determining the parents of hybrids through sequence alignment. The method described above for determining the parents of hybrids GB004-GB031 also has a high accuracy.

[0033] Example 2

[0034] (1) Since the beginning of 2023, this invention has obtained nearly a thousand maize inbred line 50K chip data. This invention attempts to construct a local database (inbred line germplasm resource database) for the protection of germplasm resources and the imitation of superior combinations of inbred lines. The urgent problem to be solved is whether the identification of maize hybrid parents can still be accurate as the amount of data in the local database increases. To this end, this invention uses the same method to remove unqualified samples (inbred line heterozygosity exceeding 3 times the standard deviation) from the local database. Using 71,246 data points from each of the nearly a thousand samples, corresponding virtual feature sequences are formed. With the help of the BLAST tool, a local database (inbred line germplasm resource database) containing 854 inbred line samples is constructed. The heterozygosity distribution diagram of the inbred lines in this embodiment is shown below. Figure 2 As shown in A, the heterozygosity analysis of some samples from 854 inbred lines is as follows. Figure 2 As shown in B.

[0035] (2) The virtual characteristic sequences of maize hybrids were compared with those in the local database (inbred line germplasm resource database). The comparison results of the GB012-GB014 hybrids of this invention in 854 inbred lines are as follows: Figure 3 As shown in (AC), Figure 3 The A-level hybrids with higher scores compared to GB012 are A420, GB094, and GB092. Figure 3 According to B, the hybrids GB013 with higher comparison scores are GB060, GB088, and GB093. Figure 3 The C-values ​​show that GB095 and GB091 are the highest-scoring hybrids of GB014. Among them, GB094 and GB092 are the parents of GB012, GB088 and GB093 are the parents of GB013, and GB095 and GB091 are the parents of GB014.

[0036] This embodiment once again verifies the accuracy of the method provided by the present invention. A420 and GB060 were matched because they have the same background as the hybrid parent (the specific inbred lines are not convenient to describe). Specifically, A420 is a sister line to GB094 and GB092, and GB060 is a sister line to GB088 and GB093.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for tracing a corn hybrid parent, comprising, The application comprises the following steps: 1) obtaining whole genome SNP loci and corresponding genotype data from a corn hybrid, screening the whole genome SNP loci and corresponding genotype data, and then sorting the whole genome SNP loci and corresponding genotype data according to chromosome number and physical position; 2) encoding the screened and sorted whole genome SNP loci corresponding genotype data into double characters to obtain a virtual characteristic sequence; the encoding process is that homozygous genotype AA is encoded as "AA", TT is encoded as "TT", and other homozygous genotypes are encoded in the same way; heterozygous genotype AT is innovatively split into double characters "A" and "T", and other heterozygous genotypes are encoded in the same way; missing data is filled with "N"; the virtual characteristic sequence is obtained by connecting the encoded double characters; 3) using a homologous alignment method to align the virtual characteristic sequence to a constructed local database, determining the corn hybrid parent according to the homologous alignment result, and realizing the traceability of the corn hybrid parent; the constructed local database comprises virtual characteristic sequences of corn inbred varieties.

2. Use of a method for tracing the origin of a parent of a hybrid corn according to claim 1, characterized in that, The application comprises any one of the following applications: 1) corn hybrid parent traceability analysis; 2) corn molecular marker assisted breeding; 3) identifying the authenticity of corn varieties; 4) corn germplasm resource protection; 5) identifying the genotype of corn hybrid offspring.

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