Maize hybrid parent traceability method and application thereof

Abstract

The invention relates to the crossing field of crop molecular breeding and biological information technology, in particular to a corn hybrid parent traceability method and application thereof. The method provided by the invention comprises the following steps: screening and sorting whole genome SNP locus data of the corn hybrid, coding the data into a virtual feature sequence, comparing the virtual feature sequence with a local database by using a homologous comparison method, and determining a hybrid parent according to a matching result. According to the method provided by the invention, the industrial problem that a common database cannot be directly utilized to accurately identify hybrid parents in the traditional technology is solved through an original data conversion mechanism and algorithm; compared with a method for identifying and tracing parents by utilizing similarity, the accuracy of a result identified by the method is greatly improved; meanwhile, the method also provides an efficient solution for variety authenticity identification, germplasm resource protection and breeding design.

Description

Technical Field

[0001] The present invention relates to the intersection of crop molecular breeding and bioinformatics technology, and specifically provides a method for tracing the parentage of corn hybrids and its application. Background Art

[0002] Common corn varieties are derived from the hybridization of two inbred lines. 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 identify superior parents. However, frequent "copycat" and "piracy" practices in the seed market seriously infringe upon breeders' rights and disrupt market order. With advances in breeding technology and growing market demand for high-quality seeds, hybrid authenticity authentication has become a core requirement for ensuring seed quality, preventing the misappropriation of germplasm resources, and curbing the circulation of counterfeit seeds.

[0003] Existing technologies mostly use molecular markers such as SNPs and INDELs and genome sequencing to trace the origin of corn samples. However, the current identification technology of corn hybrid parents has three limitations: first, molecular marker methods such as SSR and AFLP have low throughput and it is difficult to distinguish closely related materials; second, whole genome sequencing is expensive and Blast whole genome efficiency is low; third, solid phase chip analysis relies on private reference databases and cannot use public sequence resources such as NCBI.

[0004] In response to the above technical bottlenecks, the present invention innovatively proposes a path to convert the discrete genotype data of SNP sites on solid-phase chips into continuous allelic characteristic virtual sequences, breaking through the shackles of traditional analysis frameworks. Summary of the Invention

[0005] The present invention aims to provide a method for tracing the parentage of corn hybrids and its application. The core of the invention lies in establishing a technical chain of "discrete genotype → continuous feature sequence → Blast efficient matching".

[0006] The present invention provides a method for tracing the parental origin of corn hybrids, comprising the following steps: 1) Obtaining genome-wide SNP sites and corresponding genotype data of the sites from corn hybrids, screening the genome-wide SNP sites and corresponding genotype data of the sites, and then sorting the genome-wide SNP sites and corresponding genotype data of the sites according to chromosome number and physical position.

[0007] Specifically, the genotype data obtained from the maize solid-phase chip Illumina Maize SNP50 were preprocessed: high-quality SNP sites were screened (sites with a missing rate >10% and a minor allele frequency <0.05 were eliminated), and the screened SNP sites were strictly sorted according to chromosome number and physical position.

[0008] 2) encoding the genotype data corresponding to the SNP sites in the whole genome after screening and sorting into two characters, and concatenating the two characters to obtain a virtual feature sequence; Specifically, the genotype data encoding encodes the homozygous genotype AA as "AA" and TT as "TT," with other homozygous genotypes encoded similarly. The heterozygous genotype AT is innovatively split into the two characters "A" + "T," with other heterozygous genotypes encoded similarly. Missing data is padded with "N." By concatenating the two characters encoded, a double-length virtual signature sequence is generated, essentially representing the sample-specific genotype pattern.

[0009] 3) Using a homology comparison method, the virtual feature sequences are compared with a constructed local database, and the corn hybrid parents are determined according to the homology comparison results, thereby achieving traceability of the corn hybrid parents.

[0010] The local database constructed in step 3) includes virtual feature sequences of maize inbred lines. Specifically, the maize inbred lines include all maize inbred lines currently available on the market.

[0011] Specifically, when using homology alignment (Blast alignment), we used the task blastn-short mode and set the key parameter word_size=7 to accommodate short sequence matching. We also set thresholds for e-value, similarity, and sequence coverage to meet different requirements. Ultimately, the hybrid parents were determined based on the matching results, and genetically similar materials (such as sister lines) were identified through error matching analysis to provide a reference for breeding design.

[0012] The present invention provides applications of the above-mentioned method for tracing the parentage of corn hybrids, wherein the applications include any one of the following applications: 1) Analysis of parental origins of maize hybrids; 2) Maize molecular marker-assisted breeding; 3) Identify the authenticity of corn varieties; 4) Protection of corn germplasm resources; 5) Identify the genotype of corn hybrid offspring.

[0013] Compared with the prior art, the present invention has the following advantages: The method provided by the present invention converts discrete corn solid-phase gene chip site data into continuous allelic sequences that can be homologously compared through an original data conversion mechanism and algorithm, thus solving the industry problem that traditional technologies cannot directly use public databases to accurately identify hybrid parents. Compared with methods that use similarity identification and parent 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The comparison results of the hybrid GB001-GB003 of the present invention in 92 inbred lines are shown in Figure 1; A is the comparison result of the hybrid GB001, B is the comparison result of the hybrid GB002, and C is the comparison result of the hybrid GB003.

[0015] Figure 2 The figures are the heterozygosity distribution diagram of the inbred lines of the present invention and the heterozygosity analysis results of some samples among 854 inbred line samples; A is the heterozygosity distribution diagram of the inbred lines, and B is the heterozygosity analysis results of some samples among 854 inbred lines.

[0016] Figure 3 The comparison results of the hybrid GB012-GB014 of the present invention among 854 inbred lines are shown in Figure 1. A is the comparison result of the hybrid GB0012, B is the comparison result of the hybrid GB0013, and C is the comparison result of the hybrid GB0014. DETAILED DESCRIPTION

[0017] The following is a technical solution for tracing the parental origins of corn hybrids and its application, further illustrating the present invention through specific examples.

[0018] Example 1

[0019] (1) In the spring of 2023, 92 inbred lines and 31 hybrids for the combination of varieties were planted in Xinxiang, Henan Province, a corn producing area in the Huanghuaihai region. Each inbred line and hybrid was planted in one row, with a row length of 5 meters and a total of 21 plants. Before pollination, leaves from each row of materials were mixed and then sent to Wuhan Shuanglvyuan Company for DNA extraction and corn 50K chip testing. A total of 37,194 SNP loci genotype data for each of the 123 materials were obtained. Subsequently, SNP loci with poor quality (missing rate > 10% and minor allele frequency < 0.05) were removed, and finally 35,623 SNP loci genotype data for each material were obtained. The 31 hybrids and their corresponding parents are shown in Table 1 for subsequent verification of the accuracy of the results.

[0020] Table 1 Hybrid numbers and their parents 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 (2) Due to the high heterozygosity of hybrids, the results of direct similarity analysis with inbred lines are not ideal. In this embodiment, the genotype information of the SNP sites is converted into two characters after encoding. Specifically, the homozygous genotype AA is encoded as "AA", and TT is encoded as "TT", and the same applies to other homozygous genotypes; the heterozygous genotype AT is innovatively split into two characters "A" + "T", and the same applies to other heterozygous genotypes; missing data is filled with "N". By connecting the encoded two characters, a double-length virtual feature sequence is generated. In this embodiment, the encoding of 35,623 SNP sites in each of the 123 materials is converted into two characters, doubling the data to 71,246 data for each material.

[0021] (3) The 71,246 data points for each of the 92 inbred lines were concatenated and presented in FASTA format. A virtual signature sequence was generated for each inbred line. A local database was constructed using the blast software with the code makeblastdb -dbtype nucl-parse_seqids -in 92.fasta -out 92inbred. The same method was used to generate corresponding virtual signature sequences for each of the 31 hybrids. The virtual signature sequences of the 31 hybrids were then aligned in the local database with the code blastn -query F1.fasta -db 92inbred -out result.txt -outfmt7 -max_hsps 1.

[0022] (4) The comparison results of the hybrid GB001-GB003 of the present invention in 92 inbred line materials are as follows: Figure 1 (AC) shown, Figure 1 A shows that the hybrid GB001 has the highest score in the inbred lines GB067 and GB086. Figure 1 B shows that the hybrid GB002 has the highest comparison score with the inbred lines GB067 and GB087. Figure 1 C shows that the hybrid GB003 has the highest alignment score with the inbred lines GB086 and GB087, which are the male and female parents of these hybrids, verifying the high accuracy of the method provided by the present invention in determining the parents of hybrids through sequence alignment. The above sequence alignment method also has a high accuracy in determining the parents of hybrids GB004-GB031.

[0023] Example 2

[0024] (1) From the beginning of 2023 to the present, the present invention has obtained nearly a thousand 50K chip data of corn inbred lines. The present invention attempts to construct a local database (inbred line germplasm resource database) for the protection of germplasm resources and the imitation of inbred line advantage combinations. The problem that needs to be solved urgently is whether the parent judgment of corn hybrids can be accurate as the amount of data in the local database increases. To this end, the present invention adopts the same method to remove unqualified samples in the local database (the inbred line heterozygosity exceeds 3 times the standard deviation), and uses 71,246 data of each sample in nearly a thousand samples to form corresponding virtual feature sequences. With the help of blast tools, a local database containing 854 inbred line samples (inbred line germplasm resource database) is constructed. The heterozygosity distribution diagram of the inbred lines in this embodiment is shown as follows: Figure 2 As shown in A, the heterozygosity analysis of some samples in 854 inbred lines is as follows Figure 2 As shown in B.

[0025] (2) The virtual feature sequences of maize hybrids were compared with the virtual feature sequences in the local database (inbred line germplasm resource database). The comparison results of the GB012-GB014 hybrid of the present invention in 854 inbred lines are as follows: Figure 3 (AC) shown, Figure 3 A shows that the hybrid GB012 has higher scores than A420, GB094, and GB092. Figure 3 B shows that the hybrid GB013 has higher scores than GB060, GB088, and GB093. Figure 3 C shows that the hybrid GB014 has higher comparison scores with GB095 and GB091, among which 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.

[0026] This example once again verifies the accuracy of the method provided by the present invention. A420 and GB060 were compared because they have the same background as the hybrid parents (the specific inbred lines are inconvenient to describe). Specifically, A420 is a sister line to GB094 and GB092, and GB060 is a sister line to GB088 and GB093.

[0027] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for tracing the parentage of corn hybrids, characterized in that: The following steps are involved: 1) obtaining whole-genome SNP sites and corresponding genotype data of the sites from corn hybrids, screening the whole-genome SNP sites and corresponding genotype data of the sites, and then sorting the whole-genome SNP sites and corresponding genotype data of the sites according to chromosome number and physical position; 2) encoding the genotype data corresponding to the SNP sites in the whole genome after screening and sorting into two characters, and concatenating the two characters to obtain a virtual feature sequence; 3) Using a homology comparison method, the virtual feature sequences are compared with a constructed local database, and the corn hybrid parents are determined according to the homology comparison results, thereby achieving traceability of the corn hybrid parents; the constructed local database includes virtual feature sequences of corn inbred line varieties.

2. The use of the method for tracing the parentage of corn hybrids according to claim 1, characterized in that: The application includes any of the following applications: 1) Analysis of parental origins of maize hybrids; 2) Maize molecular marker-assisted breeding; 3) Identify the authenticity of corn varieties; 4) Protection of corn germplasm resources; 5) Identify the genotype of corn hybrid offspring.

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