Populus hybrid parent source identification method, system and equipment based on single-copy gene sequence and medium

Abstract

The invention belongs to the technical field of gene sequencing, and particularly relates to a populus hybrid parent source identification method, system, equipment and medium based on a single-copy gene sequence, the method comprises the following steps: firstly, constructing a single-copy gene sequence reference library containing five populus reference species, and each sequence comprises a gene coding region and upstream and downstream extension sequences thereof; secondly, acquiring second-generation sequencing data of a to-be-detected sample, comparing and assembling by taking the representative species sequence as reference, and reconstructing a single-copy gene sequence of the to-be-detected sample; and finally, determining a parent source through sequence alignment and statistical analysis. According to the method disclosed by the invention, a single-copy gene sequence can be accurately reconstructed from a hybrid sample through an optimally designed sequence assembly and comparison process, so that the coverage degree and the resolution ratio of a genetic marker are remarkably improved; in addition, a comparison screening rule and a layering judgment standard based on a complete matching length are designed, and precise identification of hybrid parent sources is realized by quantitatively analyzing the matching proportion of each reference species.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene sequencing, and particularly relates to a Populus hybrid parent source identification method, system, device and medium based on single-copy gene sequences. BACKGROUND

[0002] As important forest resources, Populus plants have long relied on morphological markers and limited molecular markers for hybrid breeding and germplasm identification. Currently, Populus classification is mainly based on morphological differences of five major clades (green poplar, white poplar, black poplar, poplar and large leaf poplar). With the development of molecular biology, existing technologies have used ITS sequences, chloroplast genomes and other molecular markers for species identification, and some studies have attempted to use SSR or SNP markers to analyze genetic relationships. In recent years, the popularity of second-generation sequencing technology has made it possible to analyze whole genome data. Some studies have developed molecular markers based on reduced genome sequencing (such as GBS) or transcriptome data. In the field of parent identification, conventional methods often use a limited number of polymorphic sites for statistical analysis, or use chloroplast sequences to trace maternal genetic information.

[0003] However, the existing parent identification methods have three key limitations: first, the resolution based on morphology or single molecular markers (such as ITS) is insufficient to distinguish closely related species or hybrid offspring; second, the chloroplast genome only reflects maternal genetic information and cannot simultaneously identify both parents; third, there is a lack of a systematic reference database of Populus single-copy genes, resulting in low throughput and poor reproducibility of existing analysis methods. Especially for Populus plants with frequent natural hybridization, existing technologies cannot accurately quantify the genetic contribution of different parents in hybrid individuals, severely restricting breeding efficiency evaluation and germplasm resource protection. These defects make it an urgent need to develop a high-precision, high-throughput parent tracing method. SUMMARY

[0004] The purpose of the present application is to provide a Populus hybrid parent source identification method, system, device and medium based on single-copy gene sequences, to solve the problem of insufficient resolution, inability to distinguish closely related species, and only reflecting maternal genetic information in the existing Populus hybrid identification method based on morphology or single molecular markers, resulting in inaccurate identification of the parent sources of hybrids.

[0005] The present application achieves the above-mentioned purpose through the following technical solutions: In a first aspect, the present application provides a Populus hybrid parent source identification method based on single-copy gene sequences, which comprises: constructing a single-copy gene sequence reference library containing reference species of each clade of Populus, each sequence containing a gene coding region and its upstream and downstream extension sequences; obtaining sequencing data of the to-be-tested Populus sample, aligning the sequencing data with reference sequences of the reference sequence library of Populus single-copy gene sequences, extracting valid reads, and assembling to reconstruct single-copy gene sequences of the to-be-tested Populus sample; performing sequence alignment on the single-copy gene sequences of the to-be-tested Populus sample and the sequence reference library, determining the matching frequency and proportion of each reference species according to the alignment result, and judging the parent source according to the matching frequency and proportion; The sequence alignment includes: analyzing characters in the alignment result and calculating the length of complete match, and when the difference between the lengths of complete match of a first alignment result and a second alignment result is greater than a preset value, the first alignment result is retained, otherwise all alignment results of the same query sequence are removed.

[0006] Further, the construction of the reference sequence library of Populus single-copy gene sequences includes: With a pre-selected Populus species genome as a reference, performing genome integrity evaluation and single-copy orthologous gene inference analysis, and taking an intersection of gene numbers of single-copy genes from the analysis results to screen single-copy orthologous genes; aligning the screened single-copy orthologous genes with other Populus species genomes, and screening single-copy sequences conserved in Populus according to alignment rate, coverage, and the like as indexes; extending each of the upstream and downstream of the coding region of the conserved single-copy sequence by a preset length to construct a sequence reference library containing multiple Populus species.

[0007] Further, the obtaining of the sequencing data of the to-be-tested Populus sample, and the aligning of the sequencing data with reference sequences in the reference sequence library of Populus single-copy gene sequences includes: preparing a second-generation sequencing library of the to-be-tested Populus sample and performing sequencing to obtain raw sequencing data; performing quality control processing on the raw sequencing data using a quality control tool to obtain valid sequencing data; aligning the valid sequencing data with reference sequences using a sequence alignment tool, with reference sequences in the reference sequence library of Populus single-copy gene sequences as the reference sequences; extracting reads with a matching degree to the reference sequences reaching a preset threshold in the alignment result, and converting the reads into double-end sequencing files and single-end sequencing files.

[0008] Further, the representative species include at least one of Populus trichocarpa, Populus deltoides, Populus alba, Populus tremula, Populus euphratica, and Populus grandidentata.

[0009] Further, the extracting of valid reads and the assembling include: sequencing the valid reads in the paired-end sequencing file and the single-end sequencing file to obtain preliminary assembled sequences; performing quality filtering on the preliminary assembled sequences to retain continuous sequences meeting a preset length as final single-copy gene sequences.

[0010] Further, the determining the parent source comprises: extracting an alignment table from the sequence alignment result, and counting matching frequencies of each reference species in the alignment table; calculating matching proportions of each reference species according to the matching frequencies; arranging the alignment table in descending order based on the matching proportions of each reference species in the alignment table; determining the parent source of the Populus sample to be tested based on the matching proportions according to a preset parent determination rule; The preset parent determination rule comprises: when the matching proportion of the first reference species reaches a first threshold value, determining that the Populus sample to be tested is a pure breed of the species; when the sum of the matching proportions of the two reference species reaches a second threshold value, determining that the Populus sample to be tested is a hybrid of the two species; when the sum of the matching proportions of the two reference species is within a third threshold value range, determining that the parents of the Populus sample to be tested are hybrid offspring; when the sum of the matching proportions of the three reference species is lower than a fourth threshold value, determining that one or both of the parents of the Populus sample to be tested are not included in the sequence reference library.

[0011] Further, the determining the reference species comprises the following steps: selecting representative species of each faction of Populus, the factions comprising the Populus ussuriensis faction, the Populus davidiana faction, the Populus nigra faction, the Populus euphratica faction, and the Populus grandidentata faction; selecting at least one species with complete genome data for each faction; the single-copy gene sequences of the selected species can cover the genetic diversity of Populus; The species with complete genome data comprise at least one of Populus trichocarpa, Populus deltoides, Populus alba, Populus tremula, and Populus euphratica.

[0012] In a second aspect, the present application provides a Populus hybrid parent source identification system based on single-copy gene sequences, which is suitable for implementing the Populus hybrid parent source identification method as described above, and the system comprises: a sequence reference library construction module, configured to construct a sequence reference library comprising single-copy gene sequences of reference species of each faction of Populus, each sequence comprising a gene coding region and its upstream and downstream extension sequences; The sequencing data processing module is configured to obtain sequencing data of the Populus sample to be tested, and align the sequencing data with reference sequences represented by the species sequences in the gene sequence reference library and extract valid reads; The sequence assembly module is configured to de novo assemble the valid reads to reconstruct single-copy gene sequences of the sample to be tested. The parent analysis module is configured to perform sequence alignment of the single-copy gene sequences of the Populus sample to be tested with the sequence reference library, determine the matching frequency and proportion of each reference species according to the alignment results, and determine the parent source based on the matching frequency and proportion. The parent analysis module includes: The matching length calculation unit is configured to parse the CIGAR string to calculate the full matching length. The alignment screening unit is configured to retain the optimal alignment when the full matching length difference of multiple alignment results of the same query sequence is greater than a preset value. The parent determination unit is configured to determine the matching frequency and proportion of each reference species according to the alignment results, and determine the parent source based on the matching frequency and proportion.

[0013] In a third aspect, the present application provides an electronic device including a memory and a processor, wherein the memory stores a computer program, and the processor implements the steps of the Populus hybrid parent source identification method as described above when executing the computer program.

[0014] In a fourth aspect, the present application provides a computer readable storage medium storing a computer program, wherein the computer program is executed by a processor to implement the steps of the Populus hybrid parent source identification method as described above.

[0015] The present application has the following advantages: 1. The present application adopts a method combining second-generation sequencing technology and bioinformatics analysis, and through an optimized sequence assembly and alignment process, the single-copy gene sequences can be accurately reconstructed from the hybrid sample, and the coverage and resolution of genetic markers are significantly improved.

[0016] 2. The present application designs an alignment screening rule and a hierarchical determination standard based on the full matching length, and through quantitative analysis of the matching proportion of each reference species, the accurate identification of the parent source of the hybrid is realized, and the limitation of the traditional method which can only make qualitative judgment is overcome.

[0017] 3. The present application constructs a single-copy gene sequence reference library covering five major factions of Populus, and systematically integrates 113 single-copy gene sequences of 24 representative species, thereby providing a comprehensive and standardized alignment basis for hybrid parent identification, and effectively solving the problem of missing reference database in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A flowchart illustrating a method for identifying the parental origin of Populus hybrids based on single-copy gene sequences, provided in one embodiment of this application; Figure 2 A schematic flowchart illustrating the construction of a Populus single-copy gene reference library provided in one embodiment of this application; Figure 3 A schematic flowchart illustrating another method for identifying the parental origin of Populus hybrids based on single-copy gene sequences, provided as an embodiment of this application; Figure 4 This is a system block diagram of a Populus hybrid parentage identification system based on a single-copy gene sequence, provided as an embodiment of this application. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] Example 1 Please see Figures 1-3 This application proposes a method for identifying the parental origin of Populus hybrids based on single-copy gene sequences in a specific embodiment. The method includes: constructing a reference library containing single-copy gene sequences of reference species from various Populus lineages, where each sequence contains a gene coding region and its upstream and downstream extension sequences; acquiring sequencing data of the Populus sample to be tested, using the sequence of a representative species in the gene sequence reference library as a reference sequence, comparing the sequencing data with the reference sequence, extracting valid reads, and assembling them to reconstruct the single-copy gene sequence of the Populus sample to be tested; comparing the single-copy gene sequence of the Populus sample to be tested with the sequence reference library, determining the matching frequency and proportion of each reference species based on the comparison results, and using this to determine the parental origin; wherein, the sequence comparison includes: parsing the characters in the comparison results and calculating the perfect match length; in multiple comparison results for the same query sequence, retaining the first comparison result when the difference in the perfect match length between the first comparison result and the second comparison result is greater than a preset value, otherwise discarding all comparison results for the query sequence.

[0021] As a preferred approach, the determination of reference species includes the following steps: selecting representative species from each lineage of the Populus genus, including the Populus alba, Populus tomentosa, Populus nigra, Populus euphratica, and Populus macrophylla; selecting at least one species with complete genome data for each lineage; the single-copy gene sequence of the selected species is sufficient to cover the genetic diversity of the Populus genus; among them, the species with complete genome data include at least one of Populus tomentosa, Populus nigra, Populus spp., Populus euphratica, and Populus euphratica.

[0022] It can be understood that the present application provides a set of high-quality single-copy gene sequence reference library covering multiple representative species of Populus and widely distributed, and reference sequences for de novo assembly of single-copy sequences of Populus species to be tested, as a basic resource for identification of genetic relationship. The reference library is obtained by screening based on existing Populus species genome assembly data, and is suitable for subsequent alignment analysis and parent tracing application.

[0023] For the core database constructed in the present application, the technical definition and characteristics are as follows: Populus SCG-RF library (Single-Copy Gene Reference Library for Populus) is a standardized single-copy gene reference sequence library, which is specially used for parent tracing analysis of Populus hybrids. The library integrates representative species genome data of five major factions of Populus, selects highly conserved single-copy gene sequences in the evolution process, and forms a reference resource after standardization.

[0024] T-SCG-RefSeq (Target-Single Copy Gene Reference Sequences) is a set of core species single-copy gene sequences selected from the complete Populus SCG-RF library, which is an optimized subset of SCG-RF library, used to guide the alignment and sequence assembly of the second-generation sequencing data of the Populus sample to be tested.

[0025] In the present application, the Populus SCG-RF library contains single-copy sequence sets of 24 species of five major factions of Populus (the white poplar faction is divided into the aspen sub-faction and the white poplar sub-faction), each species contains 113 single-copy sequences, which are widely distributed on different chromosomes of the genome, and T-SCG-RefSeq contains representative species of five major factions of Populus, i.e. Populus trichocarpa, Populus deltoides, Populus alba, Populus tremula and Populus euphratica.

[0026] In specific implementation, the second-generation sequencing data (Populus hybrid genomic DNA sequencing data obtained by second-generation high-throughput sequencing technology) of the Populus sample to be tested is used to realize the assembly of its single-copy genes through a standardized process. On this basis, the single-copy sequences assembled from the individual to be tested are subjected to multiple sequence alignment with the corresponding sequences of each species in the reference library, and are screened and counted through indicators such as complete matching length. By calculating the matching proportion of each reference species in the alignment result, the top five species with the highest matching degree are selected as candidate parent sources, so as to realize accurate identification of the parent composition of the hybrid individual and quantitative analysis of the genetic relationship.

[0027] As a preferred solution, the construction of the Populus single-copy gene sequence reference library comprises: taking a pre-selected Populus species genome as a reference, performing genome integrity evaluation and single-copy orthologous gene inference analysis, taking the intersection of the gene numbers of the single-copy genes from the analysis results, and screening the single-copy orthologous genes; aligning the screened single-copy orthologous genes with other Populus species genomes to screen the single-copy sequences conserved in Populus by taking the alignment rate and coverage as indexes; and extending the upstream and downstream of the coding region of the conserved single-copy sequences by a preset length to construct a sequence reference library containing multiple Populus species.

[0028] In specific implementation, the construction method of the Populus single-copy gene reference library realizes the screening and optimization of high-precision reference sequences through a systematic three-step process. First, taking the P. trichocarpa genome as a reference, a double verification mechanism combining BUSCO (Benchmarking Universal Single-Copy Orthologs, a tool for evaluating the integrity of genome assembly and annotation based on single-copy orthologous gene database) evaluation and OrthoFinder (a high-precision orthologous gene inference tool based on gene tree) analysis is adopted: BUSCO evaluates the integrity of the genome based on a conserved single-copy orthologous gene set, and screens the gene numbers that meet the single-copy characteristics; at the same time, OrthoFinder is used to analyze the orthogroups of multiple species genome data, and strictly screen the orthogroups that only exist in a single copy in different species. After taking the intersection of the gene numbers obtained by the two methods, sequences with a length exceeding 10,000 bp are further removed to ensure the efficiency of subsequent analysis. This step significantly improves the reliability of single-copy gene screening through cross-validation, as shown in the examples, and finally obtains a set of single-copy genes in P. trichocarpa that meet the requirements.

[0029] In the conservation verification stage, the preliminarily screened P. trichocarpa single-copy sequences are used as a reference to perform multi-sequence alignment with other Populus species genomes using minimap2 (a universal sequence fast alignment tool based on seed-extension algorithm). This step sets strict alignment rate and coverage thresholds (such as setting the requirement that the alignment rate is >90%), and screens the conserved single-copy sequences that exist universally in Populus factions to form a single-copy sequence position information set (Basic SCG set) of multiple Populus species. This cross-species conservation verification effectively solves the problem of gene introgression interference caused by natural hybridization, and ensures that the selected sequences have stable phylogenetic signals.

[0030] Based on the single copy sequence position information of multiple species of Populus, the application constructs two functionally differentiated reference libraries: for the SCG-RF library, the extension of ±1500 bp of the gene region is designed, and sufficient variation sites are provided by increasing the flanking non-coding region to significantly improve the ability to distinguish between closely related species; and the T-SCG-RefSeq selects six representative species (covering five major factions), and uses a shorter extension of ±1000 bp to optimize the calculation efficiency while ensuring assembly accuracy.

[0031] For example, the detailed information of the Populus SCG-RF library constructed by the application is shown in Table 1 and Table 2, wherein Table 1 shows all species, their factions and sequence numbers contained in the Populus SCG-RF library, and Table 2 shows the distribution of single copy sequences on chromosomes for each species contained in the Populus SCG-RF library, taking P. ussuriensis as an example.

[0032] Table 1 ; Table 2 ; As a preferred solution, the sequencing data of the Populus sample to be tested is obtained, and the sequencing data is compared with the reference sequence in the gene sequence reference library, including: preparing a next-generation sequencing library of the Populus sample to be tested and performing sequencing to obtain raw sequencing data; using a quality control tool to perform quality control processing on the raw sequencing data to obtain effective sequencing data; using the sequence alignment tool to compare the effective sequencing data with the reference sequence in the gene sequence reference library; extracting the reads in the comparison result that match the reference sequence to a preset threshold, and converting them into double-end sequencing files and single-end sequencing files. The representative species include at least one of P. trichocarpa, P. deltoides, P. alba, P. tremula, P. fremontii and P. grandidentata.

[0033] As a preferred solution, after extracting the effective reads, assembling includes: performing sequence splicing on the effective reads in the double-end sequencing files and single-end sequencing files to obtain preliminary assembly sequences; performing quality filtering on the preliminary assembly sequences to retain continuous sequences meeting a preset length as final single copy gene sequences.

[0034] According to the above embodiment, the application provides a method for constructing single copy gene sequences of a Populus species to be tested using next-generation sequencing data, which, as an example, includes the following steps when implemented: (1) Sequencing data obtaining and alignment: prepare the total DNA library of the sample to be tested, and obtain sequencing data by sequencing. After quality control of the sequencing data obtained by Trimmomatic (short read sequencing data quality control trimming software), use the T-SCG-RefSeq provided by the method as a reference, use Bowtie2 software (short read sequence rapid alignment tool) to extract successfully aligned reads (i.e. read refers to short DNA / RNA fragment sequence generated by high-throughput sequencing (NGS), representing the local genetic information of the sample to be tested), and further extract the alignment results into paired-end (Paired-end, PE, paired-end file from both ends of the DNA fragment) and single-end (Single-end, SE, file containing only single-end sequencing reads of the DNA fragment) fastq (standard text file format for storing biological sequencing data and its quality score) file by Samtools software (software for processing high-throughput sequencing NGS alignment result file).

[0035] (2) Single copy sequence assembly and quality control: use SPAdes software (short read-based de novo genome assembly software) to perform de novo assembly on the above fastq file, and filter low-quality contigs with a sequence length greater than 250 bp as a threshold to construct a single copy gene library of the contigs (i.e. gapless DNA continuous sequence spliced from overlapping reads) level of the sample to be tested.

[0036] As a preferred solution, determining the parent source includes: extracting an alignment table from the sequence alignment result, counting the matching frequencies of each reference species in the alignment table; calculating the matching proportions of each reference species according to the matching frequencies; arranging the alignment table in descending order based on the matching proportions of each reference species in the alignment table; determining the parent source of the sample to be tested based on the matching proportions according to a preset parent determination rule.

[0037] Among them, the preset parent determination rule includes: when the matching proportion of the first reference species reaches a first threshold value, it is determined that the sample to be tested is a pure breed of the species; when the sum of the matching proportions of the two reference species reaches a second threshold value, it is determined that the sample to be tested is a hybrid of the two species; when the sum of the matching proportions of the two reference species is within a third threshold range, it is determined that the parents of the sample to be tested are hybrid offspring; when the sum of the matching proportions of the three reference species is lower than a fourth threshold value, it is determined that one or both parents of the sample to be tested are not included in the sequence reference library.

[0038] As an example, in the step of judging the parent source, the single copy sequence of the sample to be tested is aligned with the Populus SCG-RF library provided by the method using minimap2, the complete matching length of each alignment is calculated by analyzing the CIGAR string, and the following filtering rules are set: if the difference between the complete matching lengths of the first alignment and the second alignment of the same query (i.e. the sequence to be queried) is greater than 10 bp, the first alignment is retained as the best alignment result, and if the difference between the complete matching lengths of the first alignment and the second alignment of the same query is less than 10 bp, all alignment results of the current query are deleted. As in Table 1, the complete matching length of the first row is the sum of all numbers before "=" in the third column CIGAR string.

[0039] As an example, in the step of judging the parent source, the single copy sequence of the sample to be tested is aligned with the Populus SCG-RF library provided by the method using minimap2, the complete matching length of each alignment is calculated by analyzing the CIGAR string, and the following filtering rules are set: if the difference between the complete matching lengths of the first alignment and the second alignment of the same query (i.e. the sequence to be queried) is greater than 10 bp, the first alignment is retained as the best alignment result, and if the difference between the complete matching lengths of the first alignment and the second alignment of the same query is less than 10 bp, all alignment results of the current query are deleted. As in Table 1, the complete matching length of the first row is the sum of all numbers before "=" in the third column CIGAR string.

[0040] More specifically, combined with the above-mentioned preset parent determination rules, for example, if the proportion value of the first alignment exceeds 0.8, it can be considered as a pure breed of the first species. For example, the first is less than 0.8, and the sum of the proportion values of the first and second alignments exceeds 0.8, then it is considered as a hybrid of the first and second species. For example, the first is less than 0.8, and the sum of the proportion values of the first and second is between 0.7-0.8, then it is considered that one of the parents is a hybrid, and the top three are considered as the main parents of the species to be tested. For example, the sum of the proportions of the top three is less than 0.7, then it is considered that one or both of the main parents is not in the library, and the top three are considered as the secondary parents, and the approximate pedigree can also be determined.

[0041] Example 2 Please refer to Figure 4, based on the same inventive concept, the present application proposes a Populus hybrid parent source identification system based on single-copy gene sequences in a specific embodiment, which is suitable for implementing the Populus hybrid parent source identification method as in Embodiment 1. The system includes a sequence reference library construction module, a sequencing data processing module, a sequence assembly module, and a parent analysis module. The sequence reference library construction module is used to construct a single-copy gene sequence reference library containing reference species of each faction of Populus, each sequence containing the coding region of the gene and its upstream and downstream extension sequences. The sequencing data processing module is used to obtain the sequencing data of the Populus sample to be tested, with the representative species sequence in the gene sequence reference library as the reference sequence, and the sequencing data is aligned with the reference sequence and the effective read is extracted. The sequence assembly module is used to de novo assemble the effective reads to reconstruct the single-copy gene sequence of the sample to be tested. The parent analysis module is used to align the single-copy gene sequence of the Populus sample to be tested with the sequence reference library, determine the matching frequency and proportion of each reference species according to the alignment result, and judge the parent source accordingly.

[0042] The parent analysis module includes a matching length calculation unit, an alignment screening unit, and a parent determination unit. The matching length calculation unit is used to analyze the CIGAR string to calculate the complete matching length. The alignment screening unit is used to retain the optimal alignment when the difference in complete matching length of multiple alignment results of the same query sequence is greater than a preset value. The parent determination unit is used to determine the matching frequency and proportion of each reference species according to the alignment result, and judge the parent source accordingly.

[0043] The specific limitations of the Populus hybrid parent source identification system based on single-copy gene sequences can be referred to the limitations of the Populus hybrid parent source identification method based on single-copy gene sequences in the above, which will not be repeated here. It should be noted that each module in the above identification system corresponds to each step in the implementation of the above identification method, and the instances and application scenarios realized by the multiple modules and the corresponding steps are the same, but are not limited to the content disclosed in Embodiment 1.

[0044] According to the above-mentioned embodiments, the working principle of the present application is that the parent source identification system of the Populus hybrid species provided by the present application is based on the conservation and specificity of single copy gene sequences, and precise identification is realized through modular design. The system is first integrated with representative species of five Populus factions by the sequence reference library construction module, and single copy orthologous genes are obtained through double screening of BUSCO and OrthoFinder. After verifying the conservation through minimap2 cross-species alignment, the reference library containing the coding region and the upstream and downstream extension sequences of the genes is constructed. After data quality control by Trimmomatic, the sequencing data of the sample to be tested is aligned with the reference sequences of the representative species by Bowtie2, and the effective reads are extracted. The sequence assembly module realizes de novo assembly by SPAdes to generate high-quality single copy gene sequences. The parent analysis module quantifies the complete matching length by matching the length calculation unit to analyze the CIGAR string; the alignment screening unit optimizes the alignment results based on a 10bp difference threshold; and the parent determination unit realizes precise determination from pure breed to complex hybrid background by combining the hierarchical proportion threshold with the genetic characteristics of the faction.

[0045] In another embodiment provided by the present application, an electronic device is also provided, which comprises a memory and a processor, the memory stores a computer program, and the processor realizes the steps of the Populus hybrid parent source identification method in embodiment 1 when executing the computer program.

[0046] In another embodiment provided by the present application, a computer readable storage medium is also provided, which stores a computer program, and the computer program realizes the steps of the Populus hybrid parent source identification method in embodiment 1 when executed by a processor.

[0047] In order to more clearly illustrate the present application and its advantages, the following will further explain the method provided by the present application in combination with specific implementation cases and related parts of the drawings.

[0048] Firstly, the second-generation sequencing data (SRR13324544) of Populus cathayana was obtained from a public data platform, and the data was generated by an Illumina HiSeq 4000 platform, and was double-end 150bp sequencing read length. The original data was subjected to quality control by Trimmomatic software, and the adapter sequence and low-quality reads were removed to obtain about 2.8 Gb of high-quality clean reads data.

[0049] In the single copy sequence assembly stage, the T-SCG-RefSeq constructed by the present application is used as the reference sequence library, and the clean reads are aligned to the reference sequence in the no-unal mode using Bowtie2 (version 2.4.2). The alignment results are converted into standardized fastq format files including double-end sequencing data (file_P1.fq.gz, file_P2.fq.gz) and single-end sequencing data (file_S.fq.gz) by Samtools. Subsequently, de novo assembly is performed in the careful mode using SPAdes (version 3.13.0), which significantly improves the assembly quality through a multi-k-mer value iterative strategy and an error correction algorithm. Finally, low-quality contigs are filtered by taking 250 bp as the length threshold, and a high-quality Populus simonii single copy gene sequence library (qing_yang_final_contigs.fasta) is obtained.

[0050] In the kinship identification stage, the assembled single copy gene sequences are compared and analyzed with the SCG-RF reference library provided by the present application. Minimap2 (parameters: -cx asm10 --eqx) is used for sequence alignment to generate PAF format alignment results containing CIGAR strings. The alignment results are parsed by a customized Python script to calculate the completely matched length of each alignment, and a 10 bp difference threshold screening rule is applied: for multiple alignment results of the same query sequence, if the completely matched length difference between the optimal alignment and the suboptimal alignment is greater than 10 bp, the optimal alignment is retained; otherwise, all alignment results of the query sequence are excluded. This strict alignment screening strategy effectively reduces false positive matches and ensures the reliability of the parent identification results. Finally, the number and proportion of each alignment to the species are calculated to confirm the kinship composition.

[0051] Table 3 ; The alignment results of the single copy gene sequences of the test species and the SCG-RF reference library are shown in Table 3. The first column is each single copy gene sequence of the test species to be aligned, the second column is the matched result in the library, the third column is the expanded alignment description string, "=" represents a completely matched alignment, "X" represents a mismatched alignment, "I" represents an insertion, and "D" represents a deletion, and the corresponding value is the value before the symbol. The fourth column is the completely matched length, which is the sum of the values corresponding to "=" in the third column.

[0052] Table 4 ; Conclusion analysis: Table 4 shows the top five results of genetic relationship identification of Populus cathayana. The sum of the top three ratios is 0.63, and the first comparison is Populus cathayana, and the last three are other members of the Populus cathayana lineage. It shows that the sample is a member of the Populus cathayana lineage, and one of the parents is Populus cathayana, and the other parent may come from other species within the Populus cathayana lineage. From the table, Populus cathayana is in a clear advantage (56 times, accounting for 44.44%) and occupies the first place, showing that it is the closest genetic relationship in the sample. Followed by Populus szechuanica, Populus ussuriensis, etc., which are Populus cathayana lineage species, and the proportion gradually decreases, indicating that these species may participate in distant hybridization or share part of the genetic background, but are not the main genetic source.

[0053] In summary, the sample has typical genetic characteristics of the Populus cathayana lineage, with Populus cathayana as the main genetic source, and it is speculated that it is the main parent of the individual; and the secondary parent may be a member of other species in the Populus cathayana lineage, such as Populus szechuanica or Populus ussuriensis. This provides a basis for further analyzing its genetic composition and hybridization history.

[0054] Those skilled in the art can appreciate that the units and algorithm steps of each example described in combination with the embodiments disclosed herein can be realized in electronic hardware or a combination of computer software and electronic hardware. Whether the functions are realized in hardware or software depends on the specific application and design constraints of the technical solutions. Professionals can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of the present application.

[0055] In addition, each functional module in each embodiment of the present application can be integrated in one processing unit, or each unit can exist physically, or two or more units can be integrated in one unit.

[0056] The above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can understand that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not drive the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for identifying the parental origin of a Populus hybrid based on single copy gene sequences, characterized by, The method comprises: constructing a reference library of single-copy gene sequences of reference species of each clade of Populus, each sequence comprising a gene coding region and its upstream and downstream extension sequences; obtaining sequencing data of a Populus sample to be tested, aligning the sequencing data with reference sequences in the reference library of gene sequences, extracting valid reads, and then assembling to reconstruct single-copy gene sequences of the Populus sample to be tested; aligning the single-copy gene sequences of the Populus sample to be tested with the sequence reference library, determining the matching frequency and proportion of each reference species according to the alignment results, and judging the parent source based thereon; wherein the sequence alignment comprises: analyzing characters in the alignment results and calculating the length of complete matches, and when the difference in the length of complete matches between a first alignment result and a second alignment result is greater than a preset value, the first alignment result is retained, otherwise all alignment results of the same query sequence are excluded.

2. The method according to claim 1, wherein the method is characterized by, The construction of the reference library of single-copy gene sequences of Populus comprises: using a pre-selected Populus species genome as a reference, performing genome integrity evaluation and single-copy orthologous gene inference analysis, taking the intersection of gene numbers of single-copy genes from the analysis results, and screening single-copy orthologous genes; aligning the screened single-copy orthologous genes with other Populus species genomes, and using alignment rate, coverage, etc. as indicators to screen single-copy sequences conserved in Populus; extending each of the coding region and its upstream and downstream of the conserved single-copy sequences by a preset length to construct a sequence reference library comprising multiple Populus species.

3. The method according to claim 1, wherein the method is characterized by, The obtaining of sequencing data of a Populus sample to be tested, the use of reference sequences in the reference library of gene sequences as reference sequences, and the alignment of the sequencing data with the reference sequences comprise: preparing a next-generation sequencing library of the Populus sample to be tested and performing sequencing to obtain raw sequencing data; performing quality control processing on the raw sequencing data using a quality control tool to obtain valid sequencing data; using sequence alignment tools to align the valid sequencing data with the reference sequences using the reference sequences in the reference library of gene sequences as reference sequences; extracting reads with a matching degree to the reference sequences reaching a preset threshold in the alignment results, and converting them into paired-end sequencing files and single-end sequencing files.

4. The method according to claim 3, wherein the method is characterized by, The reference species include at least one of P. trichocarpa, P. deltoides, P. alba, P. tremula, P. euphratica, and P. angulosa.

5. The method according to claim 3, wherein the method is characterized by, The extraction of valid reads and the subsequent assembly comprise: performing sequence splicing on valid reads in the paired-end sequencing files and the single-end sequencing files to obtain preliminary assembly sequences; performing quality filtering on the preliminary assembly sequences, and retaining continuous sequences meeting a preset length as final single-copy gene sequences.

6. The method according to claim 1, wherein the method is characterized by, The judgment of the parent source comprises: extracting an alignment table from the sequence alignment results, and counting the matching frequency of each reference species in the alignment table; calculating the matching proportion of each reference species according to the matching frequency; ranking the alignment table in descending order based on the matching proportion of each reference species in the alignment table; determining the parent source of the Populus sample to be tested based on the matching proportion according to a preset parent determination rule; wherein the preset parent determination rule comprises: when the matching proportion of the first reference species reaches the first threshold value, determining that the Populus sample to be tested is pure of the species; when the sum of the matching proportions of the two reference species reaches the second threshold value, determining that the Populus sample to be tested is hybrid of the two species; when the sum of the matching proportions of the two reference species is within the third threshold value range, determining that the parents of the Populus sample to be tested are hybrid offspring; when the sum of the matching proportions of the three reference species is lower than the fourth threshold value, determining that one or both of the parents of the Populus sample to be tested is not included in the sequence reference library.

7. The method according to claim 1, wherein the method is characterized by, The determination of the reference species comprises the following steps: selecting representative species of each Populus lineage, the lineages including the Populus ussuriensis lineage, the Populus davidiana lineage, the Populus nigra lineage, the Populus euphratica lineage, and the Populus grandidentata lineage; selecting at least one species with complete genome data for each lineage; the single-copy gene sequences of the selected species can cover the genetic diversity of Populus; The species with complete genome data include at least one of Populus trichocarpa, Populus deltoides, Populus alba, Populus tremula, and Populus euphratica.

8. A system for identifying the parental origin of a Populus hybrid based on single copy gene sequences, adapted to implement the method for identifying the parental origin of a Populus hybrid according to any one of claims 1 to 7, characterized in that, The system comprises: a sequence reference library construction module for constructing a database containing single-copy gene sequences of reference species of each Populus lineage, each sequence containing a gene coding region and its upstream and downstream extension sequences; a sequencing data processing module for obtaining sequencing data of a Populus sample to be tested, using the representative species sequences in the gene sequence reference library as reference sequences, and aligning the sequencing data with the reference sequences and extracting valid reads; a sequence assembly module for de novo assembly of the valid reads to reconstruct the single-copy gene sequence of the sample to be tested; a parent analysis module for sequence alignment of the single-copy gene sequence of the Populus sample to be tested with the sequence reference library, determining the matching frequency and proportion of each reference species according to the alignment results, and determining the parent source accordingly; The parent analysis module comprises: a matching length calculation unit for analyzing CIGAR strings to calculate the complete matching length; an alignment screening unit for retaining the optimal alignment when the difference in complete matching length of multiple alignment results of the same query sequence is greater than a preset value; a parent determination unit for determining the matching frequency and proportion of each reference species according to the alignment results, and determining the parent source accordingly.

9. An electronic device, comprising: A memory and a processor, the memory storing a computer program, the processor executing the computer program to realize the steps of the Populus hybrid parent source identification method of any one of claims 1-7.

10. A computer-readable storage medium, characterized in that, A computer program is stored, and the computer program is executed by the processor to realize the steps of the Populus hybrid parent source identification method of any one of claims 1-7.

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