Molecular markers for identification of species in the complex of oligoporus vailii and their application

Abstract

The application discloses a molecular marker for identifying species of a Catenaria sp. complex and application thereof. Based on whole genome alignment and analysis of representative species of the Catenaria sp. complex, a gene with high polymorphism and near uniform evolution among the species in the complex is obtained, a single marker is used to effectively distinguish and identify the related species of the Catenaria complex, and further, a PCR amplification primer is designed based on the DNA sequence of the gene, and a related kit is developed. The application can achieve the purpose of effectively distinguishing and identifying the related species of the Catenaria complex by using a single marker phylogenetic analysis, and compared with common multi-gene phylogenetic analysis, has the advantages of low cost, simple technology and suitability for high-throughput screening.

Description

Technical Field

[0001] This invention relates to plant pathogen detection technology, and in particular to a high-resolution molecular marker for species identification of the Colletotrichum gloeosporioides complex and its application. Background Technology

[0002] The Colletotrichum gloeosporioides species complex is an important group of pathogens in the genus Colletotrichum, containing more than 50 species that are widely distributed globally and infect a large number of economic crops such as apples, pears, strawberries, tea, tobacco, mangoes, and avocados.

[0003] Within the Colletocystis complex, species differentiation is low, interspecific genetic distances are short, and morphological differences are not obvious. ITS markers alone are insufficient to effectively distinguish closely related species, and species identification relies strictly on multigene phylogenetic analysis, which is technically complex.

[0004] Commonly used species identification markers include the intergenic spacer region (ITS) of ribosomal DNA, actin, β-tubulin, glycerol triphosphate dehydrogenase (GADPH), calmodulin (CAL), chitin synthase (CHS-1), glutamine synthetase (GS), and superoxide dismutase (SOD2).

[0005] Multigene phylogenetic analysis is currently the only accurate method for identifying species in the Colletotrichum complex. Species identification using this technique requires PCR amplification of 6-8 target genes in the tested strain, followed by sequencing of the obtained target DNA fragments. Finally, phylogenetic analysis is performed using a reference strain, and species determination is based on the phylogenetic analysis results. While this technique yields reliable results, it is cumbersome, technically complex, time-consuming, and requires a high level of expertise from the analysts. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a novel gene marker with high polymorphism and constant-rate evolution, which can achieve accurate species identification through single-gene phylogenetic analysis and reduce the technical complexity of species identification.

[0007] Therefore, the species identification sequence of the Colletotrichum complex provided by this invention is the sequence shown in SEQ ID No. 1. This sequence is a gene of the species Colletotrichum fruticosa, and its homologous genes (with a homology of ≥88.3% with the sequence shown in SEQ ID No. 1) are widely present in species of the Colletotrichum complex and exhibit high polymorphism and constant-rate evolution.

[0008] Furthermore, the present invention also provides PCR amplification primers designed based on the above-mentioned gene and the results of its homologous gene alignment.

[0009] Specifically, the PCR amplification primers include primer P1-F and primer P1-R, the sequence of primer P1-F is shown in SEQ ID No. 2, and the sequence of primer P1-R is shown in SEQ ID No. 3.

[0010] In addition, this invention also provides the application of the above-mentioned genes and primers for identifying species of the Colletotrichum complex.

[0011] Relevant identification methods include: (1) Extract genomic DNA from the test strain material; (2) Using the genomic DNA of the test strain as a template, perform PCR amplification and Sanger sequencing with the above primers; (3) Based on the Sanger sequencing results, DNA sequence multiple alignment and gene phylogenetic analysis were performed with the reference species sequence, and the species classification status of the strain was determined according to the phylogenetic tree branching pattern.

[0012] Furthermore, this invention provides the application of the above primers in the preparation of a species identification kit for the Colletotrichum gloeosporioides complex.

[0013] On the other hand, the present invention also provides a species identification kit for the Colletotrichum gloeosporioides complex. The kit includes PCR amplification reagents and the aforementioned primers.

[0014] Compared with the prior art, the present invention has at least the following advantages and beneficial effects: (1) This invention provides a high-resolution gene marker with uniform evolution characteristics for the Colloidal spore complex, which can effectively distinguish closely related species by a single molecular marker, effectively making up for the shortcomings of complex multi-gene phylogenetic methods. (2) The identification steps of this invention are simple and accurate, and the cost is low. It does not require PCR amplification of multiple gene loci, which facilitates high-throughput analysis and is easy to promote. (3) This invention can be applied to high-throughput, unbiased species diversity analysis in agricultural research, and can provide important basis for field disease diversity surveys, pest detection, etc. Attached Figure Description

[0015] Figure 1 The figures show candidate molecular markers identified by genome alignment for phylogenetic analysis of the Colletotrichum gloeosporioides complex, illustrating the phylogenetic information potential analysis of candidate sites selected from the whole genome. Figure A is a scatter plot showing the relationship between BLASTn identity and phylogenetically calibrated species divergence. The correlation coefficient and the slope of the fitted line (the steepness of the line) of the linear regression reflect the clock characteristics and phylogenetic information content of the sequences, respectively. The area marked by the red rectangle contains 29 sites, which show strong linear correlation and have high species identification potential. Figure B shows the identity distribution of Colletotrichum gloeosporioides with 15 representative phylogenetic sequences. The gray curve represents the 29 candidate sites, and the thick dashed line represents the eight control markers.

[0016] Figure 2 To compare the phylogenetic resolution characteristics of the three molecular markers M12, M15, and M28 with traditional molecular markers; Figure A is a heatmap showing the homophyletic status (yes / no) of isophylogenetic isolates in the single-gene tree; Figure B shows the Likelihood / bootstrap support values ​​of the single-gene tree; Figure C is a bar chart showing the sequence length (bp) of each marker in the C. fructicola 1104-7 reference genome; Figure D is a gene tree and species tree consistency analysis, with a heatmap showing node topological consistency (Arabic numbers correspond to...). Figure 3 (Nodes in -6A), based on PhyParts topology comparison; E diagram is a heatmap showing the DNA barcoding gap of each marker; the English abbreviations in the diagram are abbreviations of well-known terms in the field.

[0017] Figure 3 To identify the three molecular markers M12, M15, and M28, a comparison was made between the traditional ApMAT and polygenic phylogenetic trees. The figure shows the construction of a maximum likelihood (ML) phylogenetic tree based on the spliced ​​sequences of 37 markers from 54 individuals (without missing data), assuming a GTRGAMMA model with 1,000 bootstrap repetitions. Figures A–D are single-gene trees constructed based on individual markers (M12, M15, M28, and ApMat), respectively. Figure E is a combined tree constructed based on 8 traditional markers (ACT, ApMat, CAL, CHS1, GAPDH, GS, SOD2, and TUB2). Figure F is a combined tree constructed based on all 37 markers (29 newly selected markers plus 8 traditional markers).

[0018] Figure 4 shows the discriminative power comparison between the genome phylogenetic tree and the molecular marker-based phylogenetic tree; Figures (A), (B), and (C) show the comparison of maximum likelihood (ML) phylogenetic trees of the whole genome, 8 genes, and 103 representative CGSC isolates constructed using M28, respectively.

[0019] Figure 5 The results are PCR amplification results for primer sets P1-F and P1-R; NTC is a template-free control.

[0020] Figure 6 shows the species identification results of molecular markers applied to field strains. The figure illustrates the phylogenetic localization of 14 CGSC isolates based on different marker combinations of 103 reference strains; (A) Figure is the maximum likelihood (ML) phylogenetic tree constructed based on the spliced ​​sequences of eight traditional markers (ACT, ApMat, CAL, CHS1, GAPDH, GS, SOD2 and TUB2), using the GTRGAMMA model with 1,000 bootstrap repetitions; (B) Figure is the phylogenetic tree based on the M28 single gene, using the same parameters. Detailed Implementation

[0021] Unless otherwise specified, the scientific and technical terms used herein are intended for understanding by one of ordinary skill in the art. The specific embodiments of the present invention are further described below with reference to the accompanying drawings and examples.

[0022] The following examples are only used to more clearly illustrate the technical solutions of the present invention, and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001).

[0023] The sequences or biological materials used in the following examples are all from publicly available sources, such as the GenBank public database and Northwest A&F University. The traditional markers involved in the following examples: ACT, GADPH, TUB2, CAL, CHS1, SOD2, GS, and ApMAT are well-known markers for identifying the Colloidalis complex. For those skilled in the art, each marker sequence is specific and unique.

[0024] Example 1: The high-resolution molecular marker for the Colchicum collodion complex provided by this invention corresponds to a 3111 bp DNA sequence in the coding region of the Colchicum collodion gene, as shown in SEQ ID No. 1. The method for obtaining and identifying this marker is described below: (1) Select 15 species from the GenBank database covering the Colletotrichum complex ( Figure 1 B represents the genomes of 78 strains from 15 related species (the horizontal axis represents the gene pool). A local BLASTn search was performed, and strains with an E value less than 1e were identified. 10 Furthermore, under the condition that the query coverage is greater than or equal to 90%, a total of 7141 protein-coding genes that exist in all lineages were screened. (2) Further linear regression analysis based on the relationship between BLASTn identity and phylogenetically calibrated species divergence was conducted, and 29 loci with strong clock characteristics and high phylogenetic information content were selected as candidate loci. The results are as follows: Figure 1 As shown; (3) Further, based on the number of haplotypes, alignment length, number of gap sites, number of variable sites, number of phylogenetic information sites, proportion of phylogenetic information sites, and nucleotide diversity statistics between the 29 candidate sites and traditional markers (ACT, GADPH, TUB2, CAL, CHS1, SOD2, GS, ApMAT), as well as the phylogenetic information content, single-gene tree performance, barcode gap, and consistency index with the species tree, three markers, M12, M15, and M28, were selected. They outperformed traditional markers in the above evaluation indicators as a whole. The results are as follows: Figure 2 As shown.

[0025] (4) Further comparison was made between the accuracy and discriminative power of constructing phylogenetic trees using the three single-gene markers M12, M15, and M28 and the multi-gene tandem method. Among them, M28 was the best, and the results are as follows: Figure 3 As shown.

[0026] (5) The phylogenetic discrimination ability of the M28 marker was further evaluated under a larger sample range and higher species coverage: a total of 103 genomes covering 41 species of the Colletotrichum complex were selected for validation. The results are shown in Figure 4. The results show that the overall tree shapes of the genome phylogenetic tree, the eight-gene phylogenetic tree and the M28 phylogenetic tree are similar. M28 did not completely lose its phylogenetic signal in the expanded CGSC sample due to the increase in sample range, but could still recover the main phylogenetic structure of CGSC well, and has good species discrimination ability.

[0027] (6) Further, multiple sequence alignment of M28 was performed on 129 isolates from the Colletotrichum complex and 41 species. Primers were designed in the conserved region of M28 (as shown in SEQ ID No. 1) and degenerate bases were introduced to improve the amplification compatibility of the primers with different species. The final upstream primer P1-F sequence was 5′-CTAGGCTCYTCCGTYGTCATCGTGAC-3′, with a length of 26 nt; the downstream primer P1-R sequence was 5′-TGCATACTTSTCAAACGCCCATTCA-3′, with a length of 25 nt.

[0028] Based on the specific amplification primer sets P1-F and P1-R described above, a nucleic acid kit for molecular identification of the Colchicum collodion complex was prepared. The PCR reagents used in the kit, including Taq DNA polymerase, Taq DNA polymerase buffer, and dNTPs, are all commercially available products.

[0029] Example 2: This example demonstrates the use of amplification primer sets P1-F and P1-R to achieve high-efficiency PCR amplification of target sites in colloidal complex species and to detect the PCR amplification products. Details are as follows: Test strains: A total of 10 strains of the colloidal complex were used in this example. All strains were isolated from apple leaves or fruits. The strain species included 3 strains of *Colletotrichum oryzae*, 3 strains of *Colletotrichum cryptosporum*, 2 strains of *Colletotrichum siamensis*, and 2 strains of *Colletotrichum oryzae*. All strains were obtained from the Fungal Research Laboratory of Northwest A&F University.

[0030] Control primers: The control amplification primer pairs in this embodiment are ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′TCCTCCGCTTATTGATATGC-3′), as well as AM-F (5′-TCATTCTACGTATGTGCCCG-3′) and AM-R (5′-CCAGAAATACACCGAACTTGC-3′), which target and amplify the ITS and ApMAT regions, respectively.

[0031] The specific steps for identifying species in the Colletocystis complex using the kit are as follows: (1) Extract genomic DNA from the tested bacterial strains; (2) DNA amplification: The PCR amplification system was as follows: 10x Easy Taq Buffer 2.5 μL, 2.5 mM dNTPs 2 μL, 10 μM forward primer (SEQ ID No. 2) 0.5 μL, 10 μM reverse primer (SEQ ID No. 3) 0.5 μL, 100 ng / μL DNA template 1.0 μL, Easy Taq DNA polymerase 0.2 μL, and ddH2O to bring the total to 25 μL. The PCR reaction program for amplifying M28 was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 65℃ annealing for 30 s (decreasing by 0.5℃ per cycle), 72℃ extension for 2 min, 20 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 5 min; the reaction programs for amplifying ITS and ApMAT were as reported in the published literature. (3) DNA detection: The PCR amplification products were subjected to agarose gel electrophoresis. The expected band size of the M28 amplified fragment was 3000 bp. The whole genome DNA of 10 strains was extracted according to the steps in the previous embodiment, and the amplification kit and agarose gel electrophoresis were used for detection. The detection results are as follows: Figure 5 As shown, the results indicate that the M28 and ITS primer pairs for all 10 strains can amplify bands clearly, while the amplification success rate of the ApMAT primer pair is only 70%, and the bands of two strains are weak. This result shows that the kit and primer pairs of the present invention can achieve efficient amplification of the M28 target gene, and the primer amplification efficiency is better than that of ApMAT.

[0032] Example 3: This embodiment illustrates an application scenario of the high-resolution molecular markers of the Colchicum spp. complex provided by the present invention in the study of plant pathogenic biological diversity.

[0033] The *Colletotrichum gloeosporioides* complex poses a serious threat to agricultural production. Its pathogens are diverse, and species identification is complex, requiring the amplification of 6-8 target genes and multigene phylogenetic analysis. This example demonstrates, based on a novel marker, that using a single marker can effectively distinguish closely related species within the *Colletotrichum gloeosporioides* complex. The specific steps are as follows: (1) Isolation and purification of strains. Leaf and fruit samples of diseased plants were collected, and pathogens were isolated and purified based on tissue isolation method. Single-spore purification was performed on the colonies to obtain pure cultures. Fourteen strains were selected for subsequent analysis. Information on the 14 strains is shown in Table 1.

[0034] Table 1. Information on strains used for the practical validation of high-resolution molecular markers for the Colletotrichum complex.

[0035] Species identification: Species identification is based on a maximum likelihood (ML) phylogenetic tree constructed from the spliced ​​sequences of eight traditional markers (ACT, ApMat, CAL, CHS1, GAPDH, GS, SOD2, and TUB2).

[0036] (2) PCR amplification and Sanger sequencing: PCR amplification was performed on 14 strains using primer sets P1-F and P1-R. The sequences were then sequenced using forward primers (SEQ ID No. 2) and reverse primers (SEQ ID No. 3). The obtained sequences were manually proofread and low-quality sequences at both ends were removed. As a control, the sequences of the genes of actin, β-tubulin, glycerol triphosphate dehydrogenase (GADPH), calmodulin (CAL), chitin synthase (CHS-1), glutamine synthetase (GS), superoxide dismutase (SOD2), and ApMAT (Apn2-MAT locus) were also amplified by PCR, sequenced by Sanger, and manually proofread using traditional methods.

[0037] (3) Phylogenetic analysis: For the amplification products of the P1-F and P1-R primer sets, the sequence information of 14 reference strains of 14 strains was downloaded from the public database. Multiple sequence alignment was performed using MAFFT software, and a maximum likelihood phylogenetic tree was constructed using RAxML software. The GTRGAMMA model was used with 1,000 bootstrap repetitions. For the control 8 genes, multiple sequence alignment was performed using the same method, and sequence splicing was performed using SequenceMatrix software. Finally, a phylogenetic tree was constructed using RAxML software with the same parameters. The phylogenetic tree results are shown in Figure 6. The results show that the average bootstrap values ​​of the traditional multi-gene tree and the novel single-gene tree of this invention are 98.3±3.4 and 98.8±2.7, respectively, which are basically close. Among the 14 isolates, 12 isolates were completely consistent in their affiliation on the two phylogenetic trees, while the two divergent strains (PGYYL0221 and HTYO222) showed very shallow differentiation branching characteristics.

[0038] Overall, single-gene trees based on the novel loci of this invention can accurately identify species in most cases. Furthermore, compared to multigene phylogenetic analysis, the high-resolution molecular markers of the Colletotrichum complex offer advantages in efficiency, cost, and application scenarios.

[0039] Taking the species identification of a single strain as an example, compared with high-resolution molecular markers, multi-gene phylogenetic analysis requires PCR amplification and sequencing of eight genes instead of one gene. Sequence proofreading and splicing take more time, and more importantly, it is difficult to carry out high-throughput analysis based on third-generation sequencing technology.

[0040] Table 2 shows the differences in time, economic cost, convenience, accuracy, and applicability to high-throughput analysis between the high-resolution molecular markers and multigene phylogenetic analysis of the Colletotrichum gloeosporioides complex of this invention.

[0041] Table 2. Technical advantages and disadvantages of high-resolution molecular markers for the Colletotrichum gloeosporioides complex compared to traditional multigene system analysis methods.

[0042] a Cost estimation explanation: The cost of the PCR kit is calculated based on the commercial kit AP111-01 from Beijing TransGen Biotech Co., Ltd., with a cost of 0.6 yuan per PCR reaction. Including consumables and agarose gel electrophoresis detection costs, the total cost is 1.0 yuan per strain. The cost of the Sanger sequencing reaction for PCR products is estimated at 20 yuan per reaction.

[0043] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

[0044] Nucleotide sequence listing in the instruction manual SEQ ID No. 1: High-resolution molecular markers of the Colchicum cytosolicus complex corresponding to the gene sequence of Colchicum cytosolicus. SEQ ID No. 2: Primer P1-F 5′-CTAGGCTCYTCCGTYGTCATCGTGAC-3′ SEQ ID No. 3: Primer P1-R 5′-TGCATACTTSTCAAACGCCCATTCA-3′

Claims

1. A gene exhibiting high polymorphism and near-uniform evolution among species in the Colletotrichum complex, characterized by: The gene sequence is the sequence shown in SEQ ID No.

1.

2. The application of the gene of claim 1 and its closely related gene sequences in identifying species of the Colletotrichum complex, wherein the gene sequence of the closely related species has a base homology of greater than or equal to 88.3% with the sequence shown in SEQ ID No.

1.

3. The PCR amplification primers for the gene sequence of claim 1 and its closely related species, wherein the gene sequence of the closely related species has a homology of 88.3% or greater with the sequence shown in SEQ ID No.

1.

4. The PCR amplification primers as described in claim 2, characterized in that, It includes primers P1-F and P1-R, the sequence of which is shown in SEQ ID No. 2 and the sequence of which is shown in SEQ ID No.

3.

5. The application of the primers described in claim 3 or 4 for identifying species in the Colletotrichum complex.

6. A method for identifying fungal strains, characterized in that, include: (1) Extract genomic DNA from the strain material to be identified; (2) Using the genomic DNA of the strain to be identified as a template, perform PCR amplification and Sanger sequencing using the primers described in claim 2 or 3; (3) Based on the Sanger sequencing results, DNA sequence multiple alignment and gene phylogenetic analysis were performed with the reference species sequence, and the species classification status of the strain was determined according to the phylogenetic tree branching pattern.

7. The application of the primers described in claim 2 or 3 in the preparation of a kit for identifying species of the Colletotrichum gloeosporioides complex.

8. A species identification kit for the Colletotrichum gloeosporioides complex, characterized in that, The kit includes PCR amplification reagents and the primers described in claim 2 or 3.

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