Fusarium specific gene segment and use thereof in rapid detection of fusarium species

Abstract

The invention provides a fusarium specific gene segment and application of the fusarium specific gene segment in rapid detection of fusarium species. In particular, the present invention provides a fusarium specific segment. Furthermore, the invention also provides a primer pair for detecting fusarium species, and the primer pair is used for amplifying the fusarium specific segment disclosed by the invention. Furthermore, the invention also provides an application of the fusarium specific segment or the primer pair provided by the invention in detecting fusarium species. Through the fusarium specific section, different species in fusarium can be accurately distinguished, and the fusarium specific section can be applied to the field of plant quarantine to realize rapid and accurate detection of fusarium species.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to the field of fungal molecular detection, and more particularly to a Fusarium-specific gene segment and its use in rapid detection of Fusarium species. TECHNICAL BACKGROUND Fusarium was formally named by German scholar Link in 1809 (Link, H. F., (1809), Gesellschaft Naturforschender Freunde zu Berlin, Magazin 3(1): 3-42), and its taxonomic research has spanned three centuries. As one of the most important plant pathogenic fungi in the world, Fusarium not only causes serious diseases of crops, but also can directly infect humans and livestock (Leslie, J. F. & Summerell, B. A., The Fusarium Laboratory Manual). According to statistics, this genus of fungi can infect almost all major food crops, and at least 81 of the 101 most economically valuable crops are their hosts. This pathogen can cause a variety of diseases throughout the life cycle of the plant, including seed rot, seedling blight, root rot, stem rot, ear rot (including grain rot), canker, systemic wilt, and leaf spot disease, etc.

[0002] Currently, the identification of Fusarium species mainly relies on morphological characteristics, biological properties and phylogenetic analysis. However, due to the significant cross between different species in phenotypic characteristics (such as pathogenicity, toxin synthesis ability), it is difficult to achieve accurate taxonomic definition by traditional methods alone (Moretti, A. N., (2009), Zbornik Matice Srpske za Prirodne Nauke, 117, 7-13).

[0003] With the application of phylogenetic concepts in the classification of Fusarium, the problems faced by traditional taxonomy (such as relying on morphological characteristics and reproductive ability) have been gradually solved. Due to its high stability and lack of influence from the developmental stage of the strain, DNA sequence-based molecular marker technology has become the core method for species identification. Currently, the molecular identification of Fusarium is mainly achieved by analyzing the sequence similarity of multiple specific gene regions.

[0004] The sequences most commonly used to distinguish Fusarium species include: part of the genomic sequence encoding translocation elongation factor 1-alpha (tef) (Wulff, E.G. et al., (2010), Environmental microbiology, 12, 649-657), beta-tubulin (tub2) (O'Donnell, K. et al., (1998), Proceedings of the National Academy of Sciences of the United States of America, 95, 2044-2049), calmodulin (O'Donnell, K et al., (2000), Mycoscience, 41, 61-78), the internal transcribed spacer (ITS1 and ITS2) within the ribosomal repeat region (Waalwijk, C. et al., (1996), Mycologia, 88, 361-368 and O'Donnell, K. & Cigelnik, E., (1997), Molecular phylogenetics andevolution, 7, 103-116), and intergenic spacer (IGS) regions (Yli-Mattila, T. & Gagkaeva, T. inMolecular Identification of Fungi (eds Youssuf Gherbawy & Kerstin Voigt) 159-177 (Springer Berlin Heidelberg, 2010)). Not all sequences are equally effective for all species. For example, the ITS region does not work well in distinguishing closely related species of Fusarium, such as Fusarium avenaceum, Fusarium arthrosproioides 、 Fusarium tricinctum 、 F. sporotrichioides and F. langsethiae species complex and its close relatives (O'Donnell, K et al., (2000), Mycoscience, 41,61-78 and Yli-Mattila et al., (2002), Mycological Research, 106, 655-669). In addition, β-tub2 has been reported in the Fusarium solani species complex ( Fusarium solanispecies complex) (Sampietro, DA et al., (2010), Fungal Biology, 114, 74-81).

[0005] In practical applications, Fusarium spp. Fusarium ) and its closely related species. Because these species have highly similar genetic backgrounds and minimal differences in morphological characteristics, traditional identification methods (such as morphological observation or analysis of cultural characteristics) often struggle to achieve accurate identification. Existing identification technologies based on multiple molecular markers (such as multi-locus sequencing) can improve accuracy, but they are cumbersome, costly, and time-consuming, making them difficult to meet large-scale testing needs. Therefore, in the field of plant quarantine and disease control, there is an urgent need to develop a high-resolution detection technology based on a single DNA marker to achieve rapid and accurate identification and classification of Fusarium species. Summary of the Invention

[0006] In order to address the technical bottlenecks of existing Fusarium molecular identification technology, such as the identification ambiguity caused by insufficient interspecific polymorphism based on ITS sequences, the increased experimental cost (conventional methods require 3-5 primer pairs) and operational complexity (multiple rounds of PCR and data analysis take >24 hours) caused by the combined detection of multiple conserved genes, and the lack of a high-throughput detection system suitable for environmental samples, an innovative solution is proposed. By constructing a new molecular marker system, the present invention achieves (1) broad coverage of a single primer pair (compatible with at least 335 Fusarium species), (2) accurate identification efficiency of a single PCR reaction (genus / species dual specificity), and (3) cross-platform scalability of detection scenarios (compatible with traditional electrophoresis detection and high-throughput sequencing analysis of environmental samples). Specifically, the present invention innovatively constructs a Fusarium-specific gene segment labeling method based on whole-genome evolutionary characteristics, further identifies Fusarium-specific gene segments, and develops an integrated detection method that combines single strain identification and complex environmental sample analysis functions by identifying the Fusarium-specific gene segments, providing a new generation of standardized tools for medical fungal identification, crop pathogen monitoring and environmental microbiome research.

[0007] In this regard, the present invention at least provides a Fusarium-specific gene segment, primers targeting the gene segment, and use thereof in the rapid detection of Fusarium species.

[0008] Specifically, the present invention includes but is not limited to the following: In one aspect, the Fusarium-specific segment according to the present application has a length of 481-1053 bp, and / or the micro-conserved element has a length of 18 bp.

[0009] In one aspect, the Fusarium-specific segment according to the present application has a length of 481-1053 bp, and / or the micro-conserved element has a length of 18 bp.

[0010] In one aspect, the Fusarium-specific segment according to the present application has a length of 481-1053 bp.

[0011] In one aspect, the Fusarium-specific segment according to the present application has a length of 481-1053 bp.

[0012] In one aspect, the Fusarium-specific segment according to the present application has a length of 481-1053 bp.

[0013] In one aspect, the micro-conserved element according to the present application has a length of 18 bp.

[0014] In another aspect, the present application provides a method for identifying a Fusarium-specific segment, comprising the following steps: (1) performing whole genome alignment on Fusarium species; (2) screening micro-conserved elements in genome sequences of Fusarium species; (3) extracting a genomic interval segment between two micro-conserved elements; and (4) performing cross-fungus genus-specific verification on the genomic interval segment extracted in step (3) to screen segments with amplification signals only within Fusarium; In one aspect, the micro-conserved element has a length of 18-25 bp, and the number of mismatched bases between the same micro-conserved element in different Fusarium species is less than or equal to 3; the genomic interval segment has a length of 300-1200 bp and is not a region of repeat sequence and transposable element annotation; preferably, the genomic interval segment has a length of 481-1053 bp, and / or the micro-conserved element has a length of 18 bp.

[0015] In one aspect, the genomic spacer segment of the present application has a length of 481-1053 bp, and / or the microconserved unit has a length of 18 bp.

[0016] In one aspect, the genomic spacer segment of the present application has a length of 481-1053 bp.

[0017] In one aspect, the genomic spacer segment of the present application has a length of 508-1053 bp.

[0018] In one aspect, the genomic spacer segment of the present application has a length of 678, 840, 481, 810, 554, 1053, 794, 569, 762, 851, 697, 665, 999 or 508 bp. Preferably, the genomic spacer segment has a length of 840, 1053, 762, 999 or 508 bp.

[0019] In one aspect, the microconserved unit of the present application has a length of 18 bp.

[0020] In one aspect, the base sequence of the 2 microconserved units of the present application is selected from the following: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; or SEQ ID NO: 27 and SEQ ID NO: 28.

[0021] In one aspect, the base sequence of the two micro-conserved units described in the present application is as follows: SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 25 and SEQ ID NO: 26; or SEQ ID NO: 27 and SEQ ID NO: 28.

[0022] In one aspect, the Fusarium-specific segment described in the present application is selected from SEQ ID NO: 29-42.

[0023] In one aspect, the Fusarium-specific segment described in the present application is selected from SEQ ID NO: 30, 34, 41 and 42.

[0024] In one aspect, the progressive alignment algorithm guided by phylogenetic tree is used in the multiple alignment of whole genome sequences in step (1) of the method of the present application.

[0025] In one aspect, the reference genome is set to F. graminearum (Fg) strain PH-1 in the multiple alignment of whole genome sequences in step (1) of the method of the present application. Fusarium graminearum

[0026] In one aspect, in step (2) of the method of the present application, a sliding window scanning is performed to screen the micro-conserved units in the genome sequences of Fusarium species.

[0027] Specifically, in step (2) of the method of the present application, the sliding window scanning is as follows: a Python script is developed based on the MAF file, the window parameters are: window width 20 bp, step 5 bp, the conservation score (Conservation Score≥0.95) of each window is counted, the micro-conserved units (MCE) of 18-25 bp are screened out, and the number of mismatched bases between adjacent MCEs is allowed to be ≤3.

[0028] In one aspect, in step (3) of the method of the present application, double MCE structures with a spacing of 300-1200 bp are identified, the corresponding genomic intervals are extracted by BedTools v2.30.0, and the regions annotated by repeat sequences (for example, by RepeatMasker v4.1.2) and transposable elements are excluded.

[0029] ​In one aspect, in step (4) of the method of the application, virtual primers are designed for the candidate segments (e.g. with Primer3 v2.5.0, parameters: Tm = 58 ± 2°C, GC% = 40-60%), cross-validated by e-PCR (e.g. e-PCR, EMBOSS v6.6.0) in a database of 1508 non-Fusarium fungal genomes (e.g. NCBI RefSeq Fungi Release 210), and the segments with amplification signal only within the Fusarium genus are selected (e.g. allowing ≤ 2 mismatches at the 3’ end of the primers).

[0030] In yet another aspect, the application provides a primer pair for detecting or identifying a Fusarium species, which amplifies the Fusarium-specific segment of the application.

[0031] In one aspect, the primer pair sequence of the application is selected from the following: SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 47 and SEQ ID NO: 48; or SEQ ID NO: 49 and SEQ ID NO: 50.

[0032] In yet another aspect, the application provides the use of the Fusarium-specific segment of the application for detecting or identifying a Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species is selected from the group consisting of as shown in Table 1.

[0033] In one aspect, the use of the application comprises the following steps: performing sequence amplification using a primer pair targeting the Fusarium-specific segment, sequencing the amplification product, and aligning the sequencing results with a set of Fusarium species-specific sequences (e.g. the set of sequences disclosed in https: / / figshare.com / articles / dataset / 1475_14_ / 29598650?file=56381243).

[0034] In one aspect, the application uses Sanger sequencing to sequence the amplification product.

[0035] In yet another aspect, the application provides the use of the primer pair of the application for detecting or identifying a Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species is selected from the group consisting of as shown in Table 1.

[0036] In one aspect, the present application detects Fusarium species by PCR amplification using a primer pair.

[0037] In one aspect, the reaction system for PCR amplification of the present application is: 25 μL reaction system including 2x mix 12.5 μL, DNA 2 μL, primer F and R each 1 μL, ultrapure water 8.5 μL.

[0038] In one aspect, the reaction conditions for PCR amplification of the present application are: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, 33 cycles, 72℃ extension for 10 min.

[0039] In one aspect, the present application detects PCR amplification product by agarose gel electrophoresis.

[0040] In one aspect, the present application determines Fusarium species by sequencing PCR amplification product and comparing the sequencing results with Fusarium species specific sequence set.

[0041] In one aspect, the present application sequences PCR amplification product using Sanger sequencing. Preferably, the raw data is filtered by Phred≥30 quality control.

[0042] In one aspect, the present application realizes analysis of Fusarium species composition and relative abundance in samples by applying the primer pair of the present application in high-throughput sequencing platform.

[0043] In one aspect, the Fusarium species of the present application is selected from the group consisting of as shown in Table 1.

[0044] In one aspect, the Fusarium species of the present application is selected from the group consisting of Fusarium oxysporum 、 Fusarium acuminatum 、 Fusarium proliferatum 、 Fusarium fujikuroi 、 Fusarium lunulosporum 、 Fusarium noneumartii 、 Fusarium madaense, Fusarium martii, Fusarium mucidum, Fusarium terricola or Fusarium aberrans.

[0045] In one aspect, the Fusarium species of the present application is selected from the group consisting of Fusarium oxysporum 、 Fusarium acuminatum 、 Fusarium proliferatum 、 Fusarium fujikuroi 、 Fusarium lunulosporum 、 Fusarium noneumartii or Fusarium madaense.

[0046] The present study has completed whole genome sequencing and integrated analysis of 335 Fusarium species, covering 81% of the total known Fusarium species. This is the first nearly complete genus-level genomic dataset in the field of fungi, which is significantly better than existing research. Based on this data resource, we successfully developed new DNA molecular markers (in particular, Fusarium species-specific gene segments, primers designed for the specific gene segments), which can not only specifically identify Fusarium, but also further specifically and accurately distinguish different species within the Fusarium genus, while existing technologies usually require multiple pairs of primers to distinguish species in the Fusarium genus. The DNA molecular markers and their targeting primers of the present application can be applied to the field of plant quarantine to achieve rapid and accurate detection of Fusarium species. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 : Design process of screening Fusarium-specific segment scheme.

[0048] Figure 2 : Display of primer 1 for Fusarium species-specific detection results.

[0049] Figure 3 : Display of primer 2 for Fusarium species-specific detection results.

[0050] Figure 4 : Display of primer 3 for Fusarium species-specific detection results.

[0051] Figure 5 : Display of primer 4 for Fusarium species-specific detection results. DETAILED DESCRIPTION

[0052] In order for those skilled in the art to better understand the present application scheme, the technical solutions of the present application will be described clearly and completely below in conjunction with specific examples. It should be pointed out that the following detailed description is exemplary and is only an embodiment of part of the present application, not all embodiments.

[0053] Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope of protection of the present application.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, which can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0055] In summary, the present embodiment is achieved by the following innovative steps: (1) based on multiple alignment of the whole genome sequences of 335 Fusarium species, screening for genus-specific nucleic acid fragments (300-1200 bp) at the junction of conserved and variable regions, which are not conserved in other fungal genomes; (2) designing degenerate primer pairs based on the multiple sequence alignment results, ensuring that the primer annealing sites are highly conserved within the Fusarium genus; (3) establishing a standardized detection process: after extracting the total DNA of the sample to be tested, specific PCR amplification is performed, and specific bands are verified by agarose gel electrophoresis; (4) Sanger sequencing of the amplification products of positive samples, and species-level identification by comparison with the self-constructed Fusarium-specific fragment database; (5) for environmental samples, it can be expanded to a high-throughput sequencing platform to accurately analyze the composition and relative abundance of Fusarium species in complex samples. The present application breaks through the limitations of traditional detection techniques, and can complete the specific identification of the Fusarium genus through a single PCR reaction, with the advantages of high detection sensitivity and specificity, convenient operation, and low cost, etc., providing an efficient technical solution for clinical diagnosis, agricultural disease monitoring, and environmental microbial research.

[0056] In one embodiment of the present application (see, for example, Figure 1 ), the design process of the Fusarium genus-specific segment screening scheme can include the following steps: I. Phylogenetic alignment of multiple Fusarium species whole genomes Input data set: integrate the whole genome FASTA files of 335 Fusarium species and their phylogenetic tree files (Newick format), where the phylogenetic tree is constructed based on a 2,722 gene multi-site analysis.

[0057] Alignment process configuration: multiple whole genome sequence alignment is performed by Progressive Cactus v2.0.3 using the progressive alignment algorithm guided by the phylogenetic tree. In the evolverFusariumSpp.txt configuration file, define the genome file path and phylogenetic topology structure, and set the reference genome as Fusarium graminearum (strain PH-1). Fusarium graminearum

[0058] Result output: generate a whole genome collinearity alignment result file (.hal format), and convert it to a multiple sequence alignment format (MAF) using the cactus-hal2maf module in the Cactus tool chain, and set the genome block size to 1 Mb to optimize the calculation efficiency.

[0059] II. Identification of conserved regions and screening of candidate fragments ​(1) Sliding window scan: Based on the MAF file of 335 species whole genome alignment, using Python script (window parameters: window width 18-25 bp, step 1 bp), the conservation score of each window (Conservation Score≥0.95) was calculated, and all 18-25 bp micro-conserved elements (MCE) were screened. The number of mismatched bases between different species in the same MCE was allowed to be ≤3.

[0060] (2) Intergenic segment selection: Identify double MCE structure with interval 300-1200 bp, use BedTools v2.30.0 to extract the sequence of the corresponding genomic region, and exclude the region with repeat sequence and transposon element annotation.

[0061] (3) Fusarium specificity-data filtering: Extract the nucleic acid sequence of the candidate segment fragment, perform homologous sequence search on the genome database covering 1508 non-Fusarium fungi, and filter the specific fragments that are only conserved in Fusarium and do not exist in other fungi.

[0062] (4) Fusarium specificity-experimental verification: Design primers for the nucleic acid sequence of the candidate segment fragment, perform PCR on Fusarium and non-Fusarium based on the primers, and all Fusarium species have bands, while non-Fusarium species have no bands.

[0063] (5) Align database to determine Fusarium species: Perform Sanger sequencing on the DNA that can appear PCR bands, and align with the database of 335 Fusarium-specific fragments to distinguish Fusarium species.

[0064] The following is an exemplary specific embodiment combined with actual operation: Example 1 Multi-species whole genome phylogenetic alignment Input data set: Integrate the whole genome FASTA file and the phylogenetic tree file (Newick format) of 335 Fusarium species (see Appendix 1), and the phylogenetic tree is constructed based on 2,722 gene multi-site analysis. Alignment process configuration: Perform multiple whole genome alignment by Progressive Cactus v2.0.3, using progressive alignment algorithm guided by phylogenetic tree. Define the path of genome file and phylogenetic topology structure in evolverFusariumSpp.txt configuration file, set the reference genome as Fusarium graminearum (F. graminearum) strain PH-1). Fusarium graminearum

[0065] Execute command: ​cactus. / js. / evolverFusariumSpp.txt. / evolverFusariumSpp.hal --maxMemory 64G --logFile cactus.log.

[0066] Result output: Generate whole genome collinear alignment result file (.hal format), convert to multiple sequence alignment format (MAF) using cactus-hal2maf module in Cactus tool chain, set genome chunk size to 1 Mb to optimize computing efficiency: cactus-hal2maf. / js evolverFusariumSpp.hal evolverFusariumSpp.maf.gz --refGenome Fusarium_graminearum_genome.fasta --chunkSize 1000000.

[0067] Example 2: Conserved region identification and candidate fragment screening (1) Sliding window scanning: Develop Python script based on MAF file (window parameters: window width 18-25 bp, step 1 bp), count the conservation score (Conservation Score ≥ 0.95) of each window, and screen out 18-25 bp micro-conserved elements (MCE). Allow the number of mismatched bases between adjacent MCEs to be ≤ 3. Table 1 below is 14 pairs of MCE.

[0068] Table 1: Sequence information of 14 pairs of MCE

[0069] (2) Interspace section selection: Identify double MCE structure with interval of 300-1200 bp, extract corresponding genome interval using BedTools v2.30.0, and exclude repeat sequence (RepeatMasker v4.1.2) and transposable element annotation region.

[0070] (3) Cross-species specificity verification: Virtual primers (Primer3 v2.5.0, parameters: Tm=58±2℃, GC%=40-60%) were designed for the candidate segments and cross-validated by electronic PCR (e-PCR, EMBOSS v6.6.0) in a genomic database covering 1508 non-Fusarium fungi (NCBI RefSeq Fungi Release 210) to screen for segments with amplification signals only within the genus Fusarium (≤2 mismatches at the 3' end of the primer were allowed). After multiple rounds of screening, 14 Fusarium-specific fragments were obtained ( Fusarium -specific signature tags, FuST), whose specific sequences are shown in Table 2.

[0071] Table 2 Species-specific segments of 14 Fusarium species

[0072]

[0073]

[0074]

[0075] Example 3 High-precision degenerate primer design and system verification (1) Target screening and primer design Based on 14 Fusarium-specific nucleic acid marker target segments (ranging in length from 481 to 1053 bp) obtained through previous whole-genome alignment, a multi-sequence joint analysis strategy was employed: multiple sequence alignment was performed using Clustal Omega 1.2.4, and regions within each marker with conservation greater than 95% were selected for primer design. Degenerate primers were designed using Primer Premier 5.0 software, with parameters set as follows: primer length 18-24 bp, GC content 40-60%, and Tm gradient within a ±2°C range. The inventors initially generated four primer sets with gradient annealing temperatures (52-60°C) based on the specific target segments 2, 6, 13, and 14 listed in Table 2 (see Table 3).

[0076] Table 3 Information of four pairs of primers

[0077] (2) Construction of experimental verification system Sample preparation: The test strains included 7 standard strains of Fusarium spp. Fusarium oxysporum (strain number 12-60-1), Fusarium acuminatum (strain number 1-380-3), Fusarium proliferatum (strain number 14-77-1), Fusarium fujikuroi(strain number 13-65-1), Fusarium lunulosporum (strain number 106), Fusarium noneumartii (strain number 20), Fusarium madaense (strain number 74)) and 1 non-Fusarium fungus ( Epicoccum nigrum (Strain No. 7-19). Test strain culture and DNA extraction: All strains were cultured on potato dextrose agar (PDA) at 25°C. After mature colonies, mycelia were harvested, washed twice with sterile water, and ground into powder in liquid nitrogen. DNA was extracted with 2% CTAB buffer and analyzed on a 1% agarose gel.

[0078] (3) PCR amplification of the target fragment: The PCR 25 μL reaction system includes 12.5 μL of 2×mix, 2 μL of DNA, 1 μL each of primers F and R, and 8.5 μL of ultrapure water; the PCR reaction conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 60 s, 33 cycles, and extension at 72°C for 10 min.

[0079] (4) Analysis of test results: The amplified products were subjected to 1% agarose gel electrophoresis. The length of the amplified products is shown in Table 1. The positive amplified products were purified with AMPure XP magnetic beads and subjected to bidirectional Sanger sequencing using an ABI 3730xl sequencer (Shanghai Sangon Biotechnology). The raw data were filtered by Phred ≥ 30 quality control.

[0080] (5) Verification results: After systematic verification, the primer pairs in Table 3 can produce amplification products of 7 Fusarium species, while 1 non-Fusarium species cannot produce amplification products (see Figure 2 The PCR amplification products were further subjected to first-generation Sanger sequencing, and the sequences obtained were aligned with the Fusarium species-specific sequence set published in https: / / figshare.com / articles / dataset / 1475__14__ / 29598650?file=56381243, enabling accurate differentiation of seven Fusarium species (Table 4).

[0081] Table 4 Validity and accuracy of primers for identifying Fusarium species

[0082] “√” indicates the presence of PCR amplification products, and “×” indicates the absence of PCR amplification products.

[0083] Example 4 High-throughput detection technology for Fusarium colonies in environmental samples (1) Multi-functional primer design: Based on the genus-specific degenerate primers designed in the previous stage, the P5 / P7 flow cell anchor adapters compatible with Illumina sequencing platform were integrated through 5' end extension strategy, realizing the functional integration of amplification primer and sequencing adapter. This design enables the simultaneous completion of specific target segment amplification, sample multiplexing and sequencing library construction in a single PCR reaction.

[0084] (2) One-step multiplex PCR amplification-anchor ligation: HotStart Taq DNA polymerase (2.5 U / μL) and dNTPs (200 μM) system were used, and two-stage amplification program was set: the first stage (95℃ 5min; 95℃ 30s, 58℃ 45s, 72℃ 1min, 5 cycles) realized specific amplification of specific target segments; the second stage (95℃ 30s, 65℃ 45s, 72℃ 1min, 25 cycles) completed index sequence annealing and adapter ligation. Through gradient annealing optimization, the primer dimer formation rate was reduced to <0.5%.

[0085] (3) Magnetic bead standardization library preparation: AMPure XP magnetic beads (0.8x volume) were used for product purification, and Agilent 2100 Bioanalyzer was used to detect fragment distribution (main peak 200-350 bp), and Qubit 4.0 was used for quantification, and then the multi-sample library was mixed at equal molar concentration.

[0086] (4) Illumina paired-end sequencing and bioinformatics analysis: 2x150 bp paired-end sequencing was performed on NovaSeq 6000 platform, and the raw data was filtered by Fastp (v0.23.2) (Q20≥95%, reads with N content >5% were excluded). Effective data was aligned to the self-built Fusarium-specific fragment reference database by BWA-MEM (v0.7.17). SAMtools (v1.15) was used to calculate the coverage depth of specific target segments in each sample (≥30x was effective detection), and finally MetaPhlAn3 algorithm was used to calculate the relative abundance (confidence threshold≥0.95).

[0087] Table 1335 Fusarium Strains and Genome Information Overview

Claims

1. A Fusarium-specific segment characterized by, The Fusarium-specific segment is between two micro-conserved units in the genomic sequence and has a length of 300-1200 bp, wherein the micro-conserved units have a length of 18-25 bp, and the number of mismatched bases between the same micro-conserved unit in different Fusarium species is less than or equal to 3; wherein the Fusarium-specific segment is not a region of repeat sequence and transposable element annotation.

2. The Fusarium-specific segment of claim 1, characterized in that, The Fusarium-specific segment has a length of 481-1053 bp, and / or the micro-conserved unit has a length of 18 bp.

3. A method of identifying a Fusarium specific segment, comprising, The method comprises the following steps: (1) performing whole genome alignment on Fusarium species; (2) screening micro-conserved units in the genomic sequence of Fusarium species; (3) extracting the inter-genomic interval segment between the two micro-conserved units; and (4) performing cross-fungus genus-specific verification on the inter-genomic interval segment extracted in step (3) to screen segments with amplification signals only within the Fusarium genus; wherein the micro-conserved unit has a length of 18-25 bp, and the number of mismatched bases between the same micro-conserved unit in different Fusarium species is less than or equal to 3; the inter-genomic interval segment has a length of 300-1200 bp and is not a region of repeat sequence and transposable element annotation; preferably, the inter-genomic interval segment has a length of 481-1053 bp, and / or the micro-conserved unit has a length of 18 bp.

4. The Fusarium-specific segment according to claim 1 or 2 or the method according to claim 3, characterized in that, The base sequences of the two micro-conserved units are as follows: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17 and SEQ ID NO: 18; SEQ ID NO: 19 and SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22; SEQ ID NO: 23 and SEQ ID NO: 24; SEQ ID NO: 25 and SEQ ID NO: 26; or SEQ ID NO: 27 and SEQ ID NO:

28.

5. The Fusarium-specific segment according to claim 1 or 2 or the method according to claim 3, characterized in that, The Fusarium-specific segment is selected from SEQ ID NO: 29-42.

6. A primer pair for detecting or identifying Fusarium species, characterized in that, The primer pair amplifies the Fusarium-specific segment according to any one of claims 1, 2, 4 and 5.

7. The primer pair according to claim 6, characterized in that, The primer pair sequence is selected from the following: SEQ ID NO: 43 and SEQ ID NO: 44; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 47 and SEQ ID NO: 48; or SEQ ID NO: 49 and SEQ ID NO:

50.

8. Use of the Fusarium-specific segment according to any one of claims 1, 2, 4 and 5 for detecting or identifying Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species are selected as shown in Table 1.

9. Use according to claim 8, characterized in that, comprising the steps of performing sequence amplification using a primer pair targeting the Fusarium-specific segment, sequencing the amplification product, and aligning the sequencing result with a set of Fusarium species specific sequences to determine the Fusarium species.

10. Use of the primer pair according to claim 6 or 7 for detecting or identifying Fusarium species and / or analyzing the composition and relative abundance of Fusarium species in a sample, preferably the Fusarium species are selected as shown in Table 1.

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