CN111676306A

CN111676306A - SSR (simple sequence repeat) markers for colletotrichum gloeosporioides specificity of crops and detection kit thereof

Info

Publication number: CN111676306A
Application number: CN202010434257.3A
Authority: CN
Inventors: 谭新球; 张卓; 陈岳; 郑立敏; 刘勇; 张德咏; 欧阳超; 刘思珍; 李成刚; 史晓斌; 朱春晖; 曾泉
Original assignee: HUNAN PLANT PROTECTION INSTITUTE
Current assignee: HUNAN PLANT PROTECTION INSTITUTE
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-09-18

Abstract

The invention discloses a specific SSR marker for crop colletotrichum gloeosporioides, which is a marker based on a microsatellite DNA sequence repeat marker (Simple sequence repeat), wherein a primer of the specific SSR marker is selected from one of S1, S3, S4, S6, S7, S8 and S127 pairs of primers, and a fragment obtained by amplification is the SSR marker. The SSR marker 7 has good specificity to SSR markers, can be widely used for analyzing the genetic diversity in crop colletotrichum gloeosporioides populations, can also be used for distinguishing colletotrichum gloeosporioides from other colletotrichum gloeosporioides, and has important significance for crop resistance breeding, guidance of reasonable layout of resistant varieties and comprehensive field prevention and control.

Description

SSR (simple sequence repeat) markers for colletotrichum gloeosporioides specificity of crops and detection kit thereof

Technical Field

The invention belongs to the field of biotechnology and microbial detection, and particularly relates to a SSR (simple sequence repeat) marker for colletotrichum gloeosporioides specificity of crops and a detection kit thereof.

Background

Crop anthracnose is an important fungal disease in the crop production process, and is also the third major fungal disease in the world, and the crop anthracnose seriously threatens the safe production of crops. Because the heredity is complex and diverse, the population differentiation and variation are fast, and the sensitivity to chemical agents has obvious difference, how to quickly identify and diagnose the species or the microspecies is an important basis and theoretical basis for the control of pathogenic fungi; secondly, in the actual production, the resistance of the field resistant varieties is lost frequently, the differences of the resistant varieties in the same field are obvious, the reasons of the resistance loss or the differences of the field resistance are variety degeneration, pathogenic bacteria pathogenic variation or the difference change of population structure genetic diversity, and the like, and related problems are not researched much at present. Third, although relevant research at home and abroad mainly focuses on genetic diversity in a single population and finds that the genetic diversity is relatively complex, until now, many problems about whether the genetic diversity and the structure in a single population from the same host source are differentiated and whether the genetic diversity and the structure are the main reasons of field resistance loss and the like are not reported.

At present, many molecular marker technologies are applied to research on genetic diversity of plant pathogenic fungi, mainly including RFLP (DNA restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA marker), SSR (simple sequence repeat marker) or SSLP (simple sequence length polymorphism), AFLP (amplified fragment length polymorphism marker) and the like, and each technology has different advantages and disadvantages, so that the practical application is limited to different degrees. Particularly, detection techniques and kits for a single population from the same host source are rarely reported, and the current detection techniques for anthrax are mainly limited by genomic and bioinformatics analysis, and generally limit the classification and identification of ITS, TuB2, GSH, ACT, GPDH, HIS3 and CAL 7 genes (Diao et al, Colletotrichum species using anthrodsides of chili in China, Personia, 2017, (38): 20-37); other High-throughput accurate detection techniques have been developed more slowly, although (Nikita et al, Development of a High-Resolution Multi-Locus Microseptate Typing Method for Colletotrichum gloeosporioides, Mycobiology,2017,45(4): 401-; the detection system of APN2/MAT-IGS, GAP2-IGS and APN2 is established for Colletotrichum collectini (et al, optical markers for the identification of Colletotrichum species, Molecular genetics and Evolution, https:// doi.org/10.1016/j.ympev.2019.106694) and is obviously superior to the traditional 7-gene classification, but still can not obviously distinguish Colletotrichum collectini from the same host source.

Currently, the pepper anthracnose pathogenic bacteria reported internationally mainly include 16 species, wherein c.fioriniae (fruit anthracnose pathogen), c.fructicola (pine needle anthracnose pathogen), c.scoville i (staygosacchar anthracnose pathogen), c.truncataum (colletotrichum), c.gloeosporioides (colletotrichum gloeosporioides), c.acutatum (colletotrichum oxysporum) and the like are main prevalent dominant pathogenic bacteria in different production areas. And field sampling and detection prove that the infection is mostly complex infection, the complex infection rate reaches 60 percent, and anthrax populations in different regions have obvious difference. Although the common classification identification genes comprise 15 floras of the anthracnose blight bacteria of China, the complicated genetic relationship exists between the insides of the floras and other crop anthracnose bacteria, and the fact that the anthracnose blight bacteria of the capsicums have abundant genetic diversity among the floras is shown. According to earlier researches, the harm dominant population of the pepper producing area in China is colletotrichum gloeosporioides, but the genetic diversity in the colletotrichum gloeosporioides population is unclear.

Therefore, there is a need to develop a differentiation system, such as molecular markers, that can further differentiate colletotrichum gloeosporioides from other colletotrichum gloeosporioides and inherit diversity within colletotrichum gloeosporioides population.

Disclosure of Invention

On the basis of carrying out earlier stage research, the inventor takes colletotrichum capsici colletotrichum CSLL-11 genome as a starting point, 7 primers are obtained by screening from 42 pairs of SSR primers, a PCR product is 80-300bp, not only colletotrichum capsici colletotrichum from different host sources can be distinguished, and experimental results show that 5 pairs of primers can distinguish colletotrichum capsici colletotrichum from different host sources, but also other 2 pairs of primers colletotrichum have non-specificity; for different colletotrichum gloeosporioides on the same host, the 7 pairs of primers are found to be capable of distinguishing colletotrichum gloeosporioides infecting pepper from other different colletotrichum gloeosporioides, and have specificity.

Thus, the present invention has been completed. Therefore, the invention provides a specific SSR marker of colletotrichum gloeosporioides and a special detection kit thereof.

The SSR marker for crop colletotrichum gloeosporioides is a marker based on a microsatellite DNA sequence repeat marker (Simple sequence repeat), a primer of the specific SSR marker is selected from one of S1, S3, S4, S6, S7, S8 and S12 pairs of primers, and a fragment obtained by amplification is the SSR marker.

Further, the invention provides a specific primer pair for detecting the colletotrichum gloeosporioides, which is selected from one of S1, S3, S4, S6, S7, S8 and S12 pairs of primers. The amplification product of the specific primer, namely the SSR marker fragment, has the size of 80-300bp, and the sizes of the fragments can be distinguished by adopting TBE-PAGE electrophoresis. Of course, it can be performed by a method such as fluorescence PCR.

Still further, the present invention provides a kit for detecting colletotrichum gloeosporioides, which comprises one or more pairs of primers of S1, S3, S4, S6, S7, S8 and S12, preferably further comprises components required for PCR amplification, such as PCR amplification enzyme, buffer solution, genome extraction reagent; also included are reagents for detection, such as those used in gel electrophoresis, more specifically, reagents such as TBE-PAGE electrophoresis, and the like.

The invention also provides a method for detecting the colletotrichum gloeosporioides, which adopts one or the combination of primers selected from S1, S3, S4, S6, S7, S8 and S12 pairs to amplify the genome DNA template of a sample to be detected and detect the amplified product. The method can be used for distinguishing the genetic diversity in the colletotrichum gloeosporioides population of crops, and the genetic difference between colletotrichum gloeosporioides strains is confirmed according to the appearance or the deletion of target fragments of 7 pairs of primers. More preferably, one or more primer test samples of the primer pairs S3, S4, S7, S8 and S12 can be used for detection to determine genetic variation in colletotrichum gloeosporioides population, wherein the more the number of the pairs is used, the more the genetic diversity analysis can further improve the accuracy, because there may be some uncertain genetic mutations in colletotrichum gloeosporioides in nature, and the more the pairs of primers are used for detection, the lower the probability of the above cases. In addition, the primer pair of S6 or S1 can be used in combination with any one pair of other 5 pairs of primers of S3, S4, S7, S8 and S12, preferably 2-3 pairs, to distinguish between pepper and other host sources, because cross infection of colletotrichum gloeosporioides of different host sources often occurs in nature.

Wherein the extraction of the genome of the sample to be detected can be carried out by conventional methods known in the art, and the detection of the amplification product can be carried out by gel electrophoresis, more specifically by TBE-PAGE electrophoresis.

Preferably, the amplification annealing temperature of the detection method is 57 ℃ and more specifically, the reaction conditions are that a PCR reaction system is 25 mu L of 10 × Taq Buffer (containing Mg 2)⁺) 2.50. mu.L, 2.0. mu.L of 2.5mmol/L dNTP mix, 1.0. mu.L each of upstream and downstream primers (20. mu. mol/L), 0.5. mu.L of Taq DNA polymerase, 0.8. mu.L of template DNA (1:10 dilution), and ddH₂Filling O to 25 mu L;

and (3) PCR reaction conditions: 3min at 94 ℃, 30s at 94 ℃,45 s at 57 ℃, 30s at 72 ℃, 32 cycles, 10min at 72 ℃, and storing the amplified product at 4 ℃.

In the specific SSR marker, the SSR marker is microsatellite DNA based on Colletotrichum gloeosporioides (Colletotrichum gloeosporioides) CSLL-11 strain genome, and is characterized in that the number of times of dinucleotide repetition is 6 or more or the number of times of trinucleotide to hexanucleotide repetition is 5 or more.

The crops in the invention include, but are not limited to, crops of pepper, soybean, tea tree strawberry, apple, banana, mango and the like.

The invention also provides a method for carrying out genetic relationship analysis on crop colletotrichum gloeosporioides, wherein 7 pairs of primers are adopted to respectively detect samples to be detected, and if 5 or more than 5 pairs of primers in the 7 pairs of primers do not react, the samples are determined to be differential strains.

The experimental results of the invention show that: screening 12 pairs of SSR marker primers to obtain 7 pairs of specific SSR primers, wherein the fragment sizes of the specific SSR primers are respectively 88bp, 197bp, 208bp, 232bp, 240bp, 258bp and 267bp, and the specific SSR primers have better discrimination; through gradient PCR, the optimal PCR reaction conditions of different specific primers are optimized, and the optimal annealing temperature of the primers S1, S3, S4, S6, S7, S8 and S12 is 57 ℃.

The SSR primer specificity analysis among different pepper anthracnose strains finds that: the 7 pairs of primers have good specificity to pepper C.gloeosporioides, C.acutatum, C.brevisporum, C.capsici, C.truncatum and Fusarium (negative control), have no cross reaction with colletotrichum of different species of pepper, and can be used as a specific primer to distinguish colletotrichum gloeosporioides from other colletotrichum gloeosporioides.

SSR primer specificity analysis of colletotrichum gloeosporioides from different host sources: the colletotrichum gloeosporioides infecting pepper, rubber, strawberry and corn is taken as a research object, fusarium and colletotrichum gloeosporioides are taken as references, and the result shows that S6 and S1 have similar cross reaction to colletotrichum gloeosporioides from pepper, rubber and strawberry, and the colletotrichum gloeosporioides from different host sources cannot correctly distinguish which host peppers are infected by the colletotrichum gloeosporioides; the other 5 pairs of primers all showed good specificity and only reacted with colletotrichum gloeosporioides. Therefore, 7 pairs of detection primers are combined, so that colletotrichum gloeosporioides and other colletotrichum gloeosporioides can be effectively distinguished, and colletotrichum gloeosporioides of other hosts can be effectively distinguished.

And (3) carrying out SSR-PCR analysis on colletotrichum gloeosporioides strains by utilizing the 7 pairs of primers: the results of 55 colletotrichum gloeosporioides from Henan, Guangdong, Shandong, Chenzhou, Tazhou, Changde, Huahua and Changsha taken as research objects and CSLL11 taken as a reference strain show that: the regional differentiation characteristics of the test strains from different regional sources are not obvious, and the test strains present complex diversity; the strains in the same field are also diversified; the clustering analysis finds that 55 strains of colletotrichum gloeosporioides are respectively divided into 5 groups, wherein the

groups

2, 3 and 4 show abundant genetic diversity and contain mixed strains in different regions. Selecting differential strains to carry out biological and pathogenicity analysis, wherein the results show that the differential strains and the control strains have obvious differences in hypha growth rate, spore yield, spore size and pathogenicity; therefore, the differential strain which does not react above 5 pairs of primers in the 7 pairs of primers is determined, and the method can also be used for genetic relationship analysis of crop colletotrichum gloeosporioides.

In conclusion, the kit 7 has good specificity to SSR markers, can be widely used for analyzing the genetic diversity in crop colletotrichum gloeosporioides populations, can also be used for distinguishing colletotrichum gloeosporioides from other colletotrichum gloeosporioides, and has important significance for crop resistance breeding, guidance of reasonable layout of resistant varieties and comprehensive field prevention and control.

Wherein, the 7 pairs of primer sequences of the invention are as follows:

primer name	F：5’-3’	R：5’-3’
			S1	GCTAGCGAGTTGAACGCC	CCCATCCATCCCTGTTGA
S3	GGTTGTCGTACGTGCGTG	TCTTTTGCTGCTCCCGTC
			S4	GGCTGGAGCTTGGTCTCA	TCGTCAGGAACACAGCGA
S6	CAGTGGGCACTCCCAATC	CCCTCACACCGAGCTGAT
			S7	GGCAACTTCCATCCGGTA	TTGAAGGCCATCTCTCCG
S8	GAGCCATGGATGCTTTGG	CTTTGCCCACTGCCACTT
			S12	CTCACCCGTTCCGTCATC	GTCTCGTTTGGGTGGTGG

Drawings

FIG. 1, 62 strains of anthrax and other pathogenic fungi genome DNA agarose electrophoresis detection.

FIG. 2 shows SSR primer screening of colletotrichum gloeosporioides.

FIG. 3, SSR specificity analysis among different pepper anthracnose species (Note: M, DNA Marker DL 5000; 1, C. glucosporioides-CSLL 11; 2, C. acutatum; 3, C. brevisporum; 4, C. capsicii; 5, C. truncatum; 6, Fusarium. Note; M, DNA Marker DL 5000; 1, C. glucosporioides-CSLL 11; 2, C. acutatum; 3, C. brevisporum; 4, C. capsi; 5, C. truncatum; 6, Fusarium.).

FIG. 4 shows SSR specificity analysis of colletotrichum gloeosporioides from different host sources (note: M, DNA Marker DL 5000; 1, colletotrichum gloeosporioides; 2, colletotrichum gloeosporioides; 3, strawberry colletotrichum gloeosporioides; 4, colletotrichum zeae gloeosporioides; 5, colletotrichum gloeosporioides; 6, Fusarium.

FIG. 5, SSR-PCR detection of colletotrichum gloeosporioides in Henan, Shandong, Guangdong and Jilin areas (note: A: detection of S4 and S3 primers; B: detection of S12 and S6 primers; C: detection of S1 and S7 primers; and D: detection of S8 primers.).

FIG. 6, SSR-PCR detection of colletotrichum gloeosporioides in Zostera and Changsha (note: A: detection of primers S12, S4, S8 and S3 in Zostera; B: detection of primers S6, S1 and S7 in Zostera; C: detection of primers S12, S4, S8 and S3 in Changsha; D: detection of primers S6, S1 and S7 in Changsha).

FIG. 7, SSR-PCR detection of colletotrichum gloeosporioides in Chenzhou.

FIG. 8, SSR-PCR cluster analysis of 55 strains of colletotrichum gloeosporioides.

FIG. 9 growth rates (cm/d) of colletotrichum gloeosporioides differential strains.

FIG. 10 shows the colony morphology of colletotrichum gloeosporioides different strains.

FIG. 11 shows lesion sizes of colletotrichum gloeosporioides different strains.

FIG. 12 pathogenicity assay for differential strains.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials provided herein are preferred.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental techniques and experimental procedures used in the examples are, unless otherwise specified, conventional techniques, e.g. those not specifically indicated in the following examples, generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Materials, reagents and the like used in examples are commercially available from normal sources unless otherwise specified.

Example-design and Synthesis of specific SSR primers

1) MISA software (http:// pgrc. ipk-gatersleen. de/MISA /) was used based on the information on the genome of colletotrichum capsulatum C. The selection criteria were 6 or more repeats of dinucleotide and 5 or more repeats of three to six nucleotides. At the same time, SSRs interrupted in the middle by a few bases (spacing less than or equal to 100) were also screened. The operating parameters are as follows: plfilename. The output result of misa.pl is generated into a file with p3in as suffix by using a script p3in.pl, so that the primer3 calls. The operating parameter was perl p3in.pl filename.misa. Primer3 was used for batch design of primers, parameters were taken as defaults, run parameters: primer3_ core-default _ version2-output ═ filename.p3out filename.p3in. Extracting the file generated by the primer3 by using a script p3_ out.pl to obtain a final result file. perl p3out.pl filename.p3out filename.misa. And obtaining 213 correspondences by total analysis, and selecting 12 pairs of primers for screening and carrying out experimental verification based on the fragment discrimination and the repetition times of 6 nucleotides. Specific SSR primer sequences are shown in table 1 below.

2) Primer screening, using genome DNA of colletotrichum gloeosporioides CSLL11 as template, and PCR reaction system (25 μ L) 10 × Taq Buffer (containing Mg)²⁺)2.5μL，2.5mmol/2.0. mu.L of dNTP Mix, 1.0. mu.L of each of the upstream and downstream primers (20. mu. mol/L), 0.5. mu.L of Taq DNA polymerase, 0.8. mu.L of template DNA (1:10 dilution), and filling up to 25. mu.L of ddH 2O; and (3) PCR reaction conditions: 3min at 94 ℃, 30s at 94 ℃,45 s at annealing temperature (55-65 ℃), 30s at 72 ℃, 32 cycles, 10min at 72 ℃, and 4 ℃ for storage, performing gradient PCR detection, detecting PCR products by 2% agarose gel electrophoresis, and determining the optimal reaction annealing degree and the optimal working conditions of 12 pairs of primers (see table 1).

Table 1, 12 pairs of candidate SSR primer sequences

Primer name	F：5’-3’	R：5’-3’	Annealing temperature Tm (. degree.C.)
				S1	GCTAGCGAGTTGAACGCC	CCCATCCATCCCTGTTGA	57
S2	GCATGCTTCCCAACCCT	GGATGAGGCGAGACGAGA						56
				S3	GGTTGTCGTACGTGCGTG	TCTTTTGCTGCTCCCGTC	57
S4	GGCTGGAGCTTGGTCTCA	TCGTCAGGAACACAGCGA	57
				S5	GCTATGTCCGTCGTCGCT	CCAGAGACTCTTCGGCCA		58
S6	CAGTGGGCACTCCCAATC	CCCTCACACCGAGCTGAT							58
				S7	GGCAACTTCCATCCGGTA	TTGAAGGCCATCTCTCCG		56
S8	GAGCCATGGATGCTTTGG	CTTTGCCCACTGCCACTT							56
				S9	CATGATCGGGACCAGGAC	AGGGCTCAAAAGGACGCT	57
S10	TCTGAATCCAGGCAAGGC	TGCTCTTCAAGCGCCTCT						56
				S11	GAGTGATACGCCATGCCC	TTGTCCTCCAAAGGCCAG	57
S12	CTCACCCGTTCCGTCATC	GTCTCGTTTGGGTGGTGG						58

EXAMPLE two test strains genomic DNA extraction and detection

According to the Tiangen plant genomic DNA extraction kit (Tangging corporation), the centrifugation speeds of step 3, step 5 and step 6 were increased to 13000rpm, with slight modifications, according to the relevant procedures, and others were unchanged. The DNA of 62 anthrax bacteria was extracted, 2. mu.L of DNA was collected, and the DNA extraction effect was checked by electrophoresis on 1% agarose gel, and photographed on a gel imager, and stored at-20 ℃ for further use (see Table 2 and FIG. 1).

TABLE 2 information about the strains of Colletotrichum used in the study

EXAMPLE three establishment of SSR-PCR detection System

Based on the results of examples one and two, the optimal primers and the optimal annealing conditions were determined using genomic DNA of anthrax bacteria to be investigated as templates in a PCR reaction system (25. mu.L) of 10 × Taq Buffer (containing Mg 2)⁺) 2.50. mu.L, 2.5mmol/L dNTP Mix 2.0. mu.L, upstream and downstream primers (20. mu. mol/L) each 1.0. mu.L, Taq DNA polymerase 0.5. mu.L, template DNA 0.8. mu.L (1:10 dilution), add ddH2O to fill 25. mu.L; PCR reactionConditions are as follows: experiments were performed at 94 ℃ for 3min, 94 ℃ for 30s, 57 ℃ for 45s, 72 ℃ for 30s, 32 cycles, 72 ℃ for 10min, and 4 ℃ storage. Detecting PCR products by 2% agarose gel electrophoresis.

CSLL11 is used as a test strain, 7 pairs of PCR primers with specificity are obtained by screening 12 pairs of primers, S1, S3, S4, S6, S7, S8 and S12PCR products are clear and have specificity, the fragment sizes of the PCR products are respectively 88bp, 197bp, 208bp, 232bp, 240bp, 258bp and 267bp, and the PCR products have better discrimination, so the 7 pairs of primers are used as follow-up tests of SSR markers (see figure 2 and table 2). Optimal annealing temperatures of the primers S1, S3, S4, S6, S7, S8 and S12 were determined to be 57 ℃ by optimizing the optimal PCR reaction conditions for the different primers by gradient PCR (see FIG. 2).

Example four TBE-PAGE electrophoretic preparation and detection

To better distinguish the SSR-PCR products, 8% polyacrylamide gel electrophoresis detection (TBE-PAGE) was used. Preparation of 8% polyacrylamide gel (70 mL): 18.9mL of 30% acrylamide, 18.9mL of ddH2037.1mL of 5xTBE, 14mL of 10% AP1mL and 40 μ L of TEMED, and stirring uniformly. Slowly adding the prepared gel solution into the gap between the two vertical glass plates, inserting a comb, taking care that no bubbles can be generated below the comb teeth in the inserting process, and standing at normal temperature until the gel is solidified.

Sample application: and (3) placing the solidified polyacrylamide gel glass plate in an electrophoresis tank, pouring 1xTBE buffer solution into the electrophoresis tank until the solution overflows, holding two ends of a comb by hands, and slowly pulling out the comb. 1 μ L of the PCR product mixture (PCR product and 10 × Loading Buffer and fluorescent dye) was added to the gel well by pipette.

Electrophoresis: after the sample application is finished, the power supply of the electrophoresis apparatus is turned on, and electrophoresis is carried out for 1h at a voltage of 120 v.

And (3) developing: after electrophoresis is finished, the power supply is turned off, the glass plate is taken out, and each piece of PAGE gel is carefully put into a glass dish filled with ddH2O for rinsing for 2-3 times; adding a proper amount of 0.2% silver nitrate into a glass dish for dyeing, and placing the glass dish on a shaking table to ensure that the glass dish is dyed uniformly (the rotating speed of the shaking table is not too high so as to avoid breaking PAGE (polyacrylamide gel electrophoresis)), wherein the dyeing lasts for about 10 minutes; after dyeing is finished, ddH2O is rinsed for 2-3 times, newly configured developing solution is poured and placed on a shaking table until DNA bands appear and are clearly visible, the developing solution is poured off, ddH2O is rinsed for 2 times, PAGE gel is gently placed on an electrophoresis viewer, and a camera is used for taking a picture for later analysis.

EXAMPLE five SSR specificity analysis between different species of pepper anthracnose

The infected capsicum C.gloosporides, C.acutum, C.brevisporum, C.capsici and C.truncatum were used as the research objects, the Fusarium strain was used as the reference strain except anthrax, the method of example two was used to extract the genome DNA of all the strains to be tested, the primers of S1, S3, S4, S6, S7, S8 and S127 were used as the target primers, and 10 × Taq Buffer (containing Mg 2) was used according to the PCR reaction system (25 μ L)⁺) 2.50. mu.L, 2.5mmol/L dNTP Mix2.0. mu.L, 1.0. mu.L each of upstream and downstream primers (20. mu. mol/L), 0.5. mu.L of Taq DNA polymerase, 0.8. mu.L of template DNA (1:10 dilution), and addition of ddH2O to 25. mu.L; and (3) PCR reaction conditions: experiments were performed at 94 ℃ for 3min, 94 ℃ for 30s, 57 ℃ for 45s, 72 ℃ for 30s, 32 cycles, 72 ℃ for 10min, and 4 ℃ storage. And (4) detecting the PCR product by TBE-PAGE electrophoresis, and specifically referring to example four. The results show that: the 7 pairs of primers have good specificity, do not react with the pepper colletotrichum of different species on the same host, and can be used as a specific primer to distinguish the colletotrichum gloeosporioides from other colletotrichum gloeosporioides (see figure 3).

Example SSR specificity analysis of colletotrichum gloeosporioides from six different host sources

The genomic DNA of all the test strains was extracted by the method of example two using colletotrichum gloeosporioides infecting capsicum, rubber, strawberry and corn as the study target and fusarium and colletotrichum as the reference, and the primers S1, S3, S4, S6, S7, S8 and S127 as the target primers according to the PCR reaction system (25. mu.L): 10 × Taq Buffer (containing Mg 2)⁺) 2.50. mu.L, 2.5mmol/L dNTP Mix 2.0. mu.L, upstream and downstream primers (20. mu. mol/L) each 1.0. mu.L, Taq DNApolymerase 0.5. mu.L, template DNA 0.8. mu.L (1:10 dilution), add ddH2O to fill 25. mu.L; and (3) PCR reaction conditions: 3min at 94 ℃, 30s at 94 ℃,45 s at 57 ℃, 30s at 72 ℃, 32 cycles, 10min at 72 ℃, and storage at 4 ℃ in sequenceAnd (5) carrying out an experiment. And (4) detecting the PCR product by TBE-PAGE electrophoresis, and specifically referring to example four. The results show that: the 5 pairs of primers showed good specificity, except that S6 and S1 showed similar responses to colletotrichum gloeosporioides from capsicum, rubber and strawberry. Therefore, the 7 pairs of primers can be used in combination to distinguish Colletotrichum gloeosporioides from different hosts (FIG. 4).

Example SSR-PCR analysis of colletotrichum gloeosporioides in strains

Statistical analysis was performed by means of manual counting according to the polyacrylamide electrophoresis pattern (DNA fragment) of TBE-PAGE nucleic acid. The principle is to count binary data with or without bands, the bands are marked with 1, and the bands are marked with 0. The bands present at the same positions, all recorded as 1, represent the binding site for one primer. DNA fingerprint spectrum of test strain is constructed according to the obtained 0-1 matrix, and the genetic relationship of 55 parts of colletotrichum gloeosporioides is analyzed (see table 2).

TABLE 2 construction of 55 parts matrix of colletotrichum gloeosporioides 0-1

The SSR-PCR reaction was carried out by using 7 pairs of primers, CSLL11, and colletotrichum gloeosporioides from Henan, Guangdong, Shandong, Chenzhou, Tazhou, Changde, Huahua and Changsha as research targets and CSLL11 as reference strains. The results show that: the regional differentiation characteristics of the test strains from different regional sources are not obvious, and the test strains present complex diversity. For example, the strains in Hunan province are most diverse, strains in Changsha, Hengyang and Chenzhou are distributed in a plurality of branches of a cluster, and strains in the same field are not clustered together; the strain diversity in Heider and Taoism areas is relatively simple, and the strain has certain area distribution characteristics. 9 test strains in Henan province have obvious regional distribution characteristics and small genetic distance difference; the 7 test strains in Shandong province were clustered in multiple branches. Interestingly, it was found that 10 strains, Cg10, Cg13, Cg14 from Shandong, CZAR21, CZXN11, CZXN12 from Chenzhou, CSLL02, CSLL13 from Changsha, and ZZHNA16, ZZPYM01 from Tahou, were significantly different from the other test strains. According to the cluster analysis result, 55 strains of colletotrichum gloeosporioides are respectively divided into 5 groups, wherein the

groups

2, 3 and 4 show abundant genetic diversity, and strains containing different regions are mixed and form, and simultaneously show a certain regional distribution characteristic. While group1 and group5 are relatively simple to compose. See fig. 5, 6, 7 and 8. Wherein, FIG. 5 shows SSR-PCR detection of colletotrichum gloeosporioides in Henan, Shandong, Guangdong and Jilin areas, and compared with a test reference strain CSLL-11, the SSR-PCR detection shows that the colletotrichum gloeosporioides in Henan, Shandong, Guangdong and Jilin areas have obvious intraspecific genetic diversity; FIG. 6 shows SSR-PCR detection of colletotrichum gloeosporioides in continents and Changsha areas, and compared with a test reference strain CSLL-11, the same findings indicate that the colletotrichum gloeosporioides in continents and Changsha areas have genetic diversity and the colletotrichum gloeosporioides in the same field of Changsha also have genetic variation; FIG. 7 and FIG. 8 show SSR-PCR detection of colletotrichum capsici colletotrichum and colletotrichum capsici colletotrichum in Chenzhou region, respectively, and similar results are verified.

EXAMPLES example application of eight SSR markers

1) Selection of differential strains

According to SSR-PCR results, combining a clustering matrix, comparing with CSLL11, determining that the non-reacted strain above 5 primers in 7 pairs of primers is a differential strain, and selecting Cg10, Cg13 and Cg14 of Shandong; chenzhou CZAR21, CZXN11, CZXN 12; 10 strains, such as CSLL02 and CSLL13 from Changsha and ZZHNA16 and ZZPYM01 from Ribosa, were used as the differential strains (see Table 3).

TABLE 3 selection of differential strains

2) Comparison of growth of different strains

The growth rate of the strain is measured by adopting a strain cake method, and a sporulation amount experiment is carried out by utilizing a shake bacteria method. The results of the experiments showed that 10 strains differed from the control strain CSLL11 in colony morphology and growth rate to a different extent (table 4). Differential strain hyphal growth rate analysis, although the average growth rates of CSLL13, Cg10, Cg13, ZZPYM01, CZXN11 and CZXN12 strains were not significantly different compared to the control strain CSLL11, their growth rates were faster than the control strain; whereas the Cg14, CSLL02, CZAR21, ZZHNA16 strains had average growth rates slower than the reference strain CSLL11 and made significant differences (see fig. 9). The colony morphology analysis of the differential strain shows that compared with a control strain CSLL11, CSLL13, Cg10, Cg13 and ZZPYM01 have relatively developed aerial hyphae and grow in radioactivity relative to CSLL 11; the colony morphology of CZAR21 and Cg14 varied relative to the other strains, with ragged colony edges (see fig. 10).

TABLE 4 Difference strains conidia morphology Difference comparison (μm)

Note: "-", not determined.

3) Determination of pathogenicity of differential strains

The pathogenicity is measured by adopting a hypha block inoculation in vitro Benshi tobacco leaf method. The results showed that CSLL02 was more pathogenic than CSLL11, the Cg10 strain was equally pathogenic than CSLL11, and the remaining strains were significantly less pathogenic than CSLL11, with CZXN11 and CZXN12 being the least pathogenic, indicating that there was significant differentiation in the differential strains pathogenicity (see figures 11 and 12). FIG. 11 is a significance analysis of lesion size data for the differential strains, with CSLL02 being significantly more pathogenic than CSLL11 and Cg10 being comparable to CSLL11 compared to the control strain CSLL11, but with CSLL02 and Cg10 being not significantly different from CSLL 11; the pathogenicity of other 8 test strains is weaker than that of CSLL11, wherein Cg13, CAXN11, ZZHNA16 and ZZPYM 014 strains have significant difference from that of CSLL11, and no association among the biological traits is found by combining sporulation quantity, conidiomorphism and hypha growth rate. FIG. 12 shows the hyphal mass inoculation and disease symptom region.

Sequence listing

<110> plant protection institute of Hunan province

<120> SSR (simple sequence repeat) marker for colletotrichum gloeosporioides specificity of crops and detection kit thereof

<160>24

<170>PatentIn Version 3.1

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Claims

1. An SSR marker for specificity of colletotrichum gloeosporioides of crops, wherein a primer of the SSR marker is selected from one of primer pairs S1, S3, S4, S6, S7, S8 and S127, and a fragment obtained by amplification is the SSR marker;

the sequences of the primers are as follows:

2. a primer pair for detecting the specificity of colletotrichum gloeosporioides is selected from one of S1, S3, S4, S6, S7, S8 and S127 pairs of primers; the sequences of the primers are as follows:

3. a kit for detecting colletotrichum gloeosporioides of crops comprises one or more pairs of primers S1, S3, S4, S6, S7, S8 and S127; the sequences of the primers are as follows:

4. the kit of claim 3, further comprising components required for PCR amplification, such as PCR amplification enzymes, buffers, genome extraction reagents; also included are reagents for detection, for example, in gel electrophoresis, more particularly using, for example, TBE-PAGE electrophoresis.

5. A method for detecting colletotrichum gloeosporioides adopts one or a combination of primer pairs selected from S1, S3, S4, S6, S7, S8 and S127 to amplify a genome DNA template of a sample to be detected and detect an amplification product;

the sequences of the primer pairs are as follows:

6. the method of claim 5, wherein the samples are tested using one or more of the primer pairs S3, S4, S7, S8, and S12 to determine genetic diversity within the colletotrichum gloeosporioides population, and further wherein the samples are tested using the primers S6 or S1 to distinguish the host source of colletotrichum gloeosporioides.

7. The method of claim 5 or 6, wherein the detection of the amplification product is performed by gel electrophoresis, more particularly by TBE-PAGE electrophoresis.

8. The method of claim 5 or 6, wherein the detection method has an amplification annealing temperature of 57 ℃.

9. The method of claim 5 or 6, wherein the amplification reaction is carried out under conditions such that the PCR reaction system contains 25. mu.L of 10 × Mg2⁺2.50 μ L of Taq Buffer, 2.0 μ L of 2.5mmol/L dNTP mixture, 1.0 μ L of each of 20 μmol/L upstream and downstream primers, 0.5 μ L Taq DNA polymerase, 0.8 μ L template DNA, and ddH₂Filling O to 25 mu L; and (3) PCR reaction conditions: 3min at 94 ℃, 30s at 94 ℃,45 s at 57 ℃, 30s at 72 ℃, 32 cycles, 10min at 72 ℃, and storing the amplified product at 4 ℃.

10. The method of claim 5 or 6, wherein the crop is selected from the group consisting of pepper, soybean, tea tree strawberry, apple, banana, mango.