KR102086735B1 - Molecular marker for selecting Xanthomonas campestris pv. campestris race 1 of Brassicaceae crops and selection method using the same molecular marker - Google Patents

Abstract

The present invention relates to a specific molecular marker capable of discriminating the Xanthomonas campestris pv. campestris race 1 of Brassicaceae crops and a method for discriminating the Xanthomonas campestris pv. campestris race 1 of Brassicaceae crops using the same. The Xanthomonas campestris pv. campestris race 1 of Brassicaceae crops is discriminated by using the molecular marker according to the present invention such that the Xanthomonas campestris pv. campestris race 1 of the Brassicaceae crops can be simply and accurately discriminated, thereby being useful for saving time, costs, and effects required for raising the Brassicaceae crops.

Description

Molecular marker for selecting Xanthomonas campestris pv. campestris race 1 of Brassicaceae crops and selection method using the same molecular marker}

The present invention relates to a black rot bacterium (Xanthomonas campestris pv. Campestris) Black rot fungus race 1 determines the baechugwa crops using specific molecular markers and that it can determine the race 1 Method of baechugwa crops, more specifically baechugwa crops The present invention relates to a molecular marker that can discriminate cabbage and crop black rot bacteria by extracting genomic DNA of a black rot bacterium, which causes serious diseases.

Baechugwa crops rot in the black by the black rot bacteria (Xanthomonas campestris pv. Campestris, ( Xcc)) is one of the most important diseases in baechugwa (Brassicaceae) crops. In addition, black rot is a disease that can cause more than 50% of economic losses from pathogen infection even under favorable plant growth conditions (Williams, PH, Plant Dis. 1980, 64: 736-7422).

Xcc is a small, rod-like, motility, aerobic, gram-negative, non-sporing bacterium (Sewariya, VK et al ., Int. J. Pharma. And Bio.Sci . 2012, 3 : 441-453) ). Pathogens are mainly transmitted through vascular tissues through drainage tissues, but also through wounded tissues and pores. These black rot bacteria prefer hot and humid environments and are rapidly transmitted by rainwater and irrigation. Infected seeds, plants, and crop debris become inoculations of black rot. Although pesticides are used to kill black rot, Xcc is resistant to fungicides, making it very difficult to control black rot. Therefore, the development of black rot resistant varieties can be said to be the most effective to control the black rot.

Xcc has been classified into 11 races to date based on their interaction with different varieties and pathogens (Kamoun et al ., Mol. Plant-Microbe Interact. 1992, 5: 22-33; Vicente et al ., Phytopa thology. 2001, 91: 492-499; Fargier and Manceau, Plant Pathol. 2007, 56: 805-818; Cruz et al ., J. Plant Pathol. 2017, 99: 403-414). Several studies have reported the presence of Xcc races in different regions. In 2001, Vincent et al. Announced the separation of six races (race 1, 2, 3, 4, 5, 6) from the United Kingdom, and seven races (race 1, 4, 5, 6, 7) in northwestern Spain. , 8 and 9), and race 4 and race 6 were superior to broccoli ( B. oleracea ) and turnip ( B. rapa ), respectively. Five races (race 1, 4, 5, 6, 7) were found in Nepal, of which race 1, 4 and 6 were the most common (Jensen et al ., Plant Dis . 2010, 94: 298-305). Three races (race 1, 4, 6) have been reported in India (Singh et al ., Seed Sci Technol. 2016, 42: 36-46) and four races in Iran (race 1, 4, 5, 6). (Rouhrazi and Khodakaramian, Eur. J. Plant Pathol . 2014, 139: 175-18) , and four races (race 1, 3, 4, 6) were isolated from South Africa (Chidamba and Bezuidenhout, Eur. J. Plant. Pathol . 2012, 133: 811-818). In 2017, two races (race 10, 11) were found in Portugal (Cruz et al ., J. Plant Pathol. 2017, 99: 403-414).

Quick and easy race- specific detection of Xcc at the molecular level is very important for the effective management of black rot. However, no race-specific molecular marker for detecting a specific Xcc race has yet been developed. Previously, a real-time PCR method for Xcc detection in Brassica seeds was developed (Berg et al ., Lett Appl Microbiol 2006, 42: 624-630). In addition, the polymerase chain reaction that is based on a repetitive DNA sequence elements (rep-PCR) is a black rot bacterium (Xanthomonas campestris pv. Campestris) of baechugwa plant, bacterial jeommunuibyeong bacteria (Xanthomonas campestri s pv. Vesicatoria), rice huinip blight bacteria (. Xanthomonas oryzae pv oryzae) and has been used to classify and identify the genomic structure of the banana blight (Xanthomonas campestris pv musacearum.) ( Cruz et al, 1996, Phytopathology 86:.. 1352-1359; Louws et al 1995, Phytopathology 85: 528-536; Cruz et al . 1996, Phytopathology 86: 1352-1359; Aritua et al . 2007, J. Biotechnol . 6: 179; Singh et al . 2011, Plant Pathol . 65: 1411-1418). However, this method is only for detecting and confirming Xcc, and in order to distinguish races of each black rot bacterium, the incidences of each black rot bacteria are inoculated after inoculating the black rot bacteria using multiple discriminating varieties / systems. The race can be determined by observation, which is not easy because it takes a lot of time and labor.

In the present invention, by developing a baechugwa crops black rot bacterium (Xcc) race 1 and the other race of the race- specific PCR-based molecular marker for rapidly detecting and identifying from the strain of using them for quick and easy Xcc race 1 The present invention has been completed by discovering that it can be detected accurately.

Therefore, the technical problem to be solved in the present invention is to provide a molecular marker for discriminating black rot bacteria race 1 of Chinese cabbage and crops.

In addition, another technical problem to be solved in the present invention is to provide a method for determining the black rot bacteria race 1 of Chinese cabbage and crops.

In addition, another technical problem to be solved by the present invention is to provide a kit for determining the black rot bacterium race 1 of Chinese cabbage and crops comprising the molecular marker.

In order to solve the above technical problem, the present invention includes a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4, black rot fungus ( Xcc ) race 1 of the Chinese cabbage crops It provides a primer set that is a molecular marker to determine the.

In addition, the present invention (S1) separating the DNA from the black rot bacteria ( Xcc ) of Chinese cabbage and crops; (S2) amplifying a target sequence by performing a polymerase chain reaction (PCR) by mixing the template isolated from the step (S1) with the primer set described above; And (S3) provides a method for determining the black rot bacteria ( Xcc ) race 1 of Chinese cabbage and crops comprising the step of separating the PCR product amplified by the step (S2) by size.

In addition, the present invention provides a kit for determining black rot bacteria ( Xcc ) race 1 of Chinese cabbage and crops comprising the primer set.

In the present invention, as a molecular marker that can distinguish Xcc race 1 of previously reported cabbage and crops, a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 (Xcc_47R1) or a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4 A primer set was developed comprising (Xcc_85R1).

Cabbage crops in the present invention includes all crops that can be infected with black rot bacteria such as cabbage, radish, cabbage.

In the context of the present invention, the term "primer" is a single strand of oligonucleotide complementary to the nucleic acid strand to be replicated, and the reagents (DNA polymerase or reverse transcriptase) and the four different dNTPs for polymerization at the appropriate buffer and temperature ( in the presence of deoxynucleoside triphosphate). Suitable lengths of the primers are determined by the properties of the primers to be used, but are typically 18 or more, preferably 20 to 200, more preferably 20 to 100, even more preferably 20 to 30 consecutive Consists of nucleotides and generally requires lower temperatures to form a base pair that is sufficiently stable with the template.

In addition, the primers may incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. Primers of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, “capsulation”, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, etc.) or charged linkages such as phosphorothioate, phosphorodithioate and the like. Nucleic acids may be selected from one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine, psoralene, etc.). ), Chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg horseradish oxidase, alkaline phosphatase), radioisotopes (eg ³² P), fluorescent molecules, chemical groups (eg biotin), and the like.

In another aspect, the present invention relates to a method for screening Xcc race 1 of Chinese cabbage crops, comprising the following steps:

(S1) separating the DNA from the black rot strains of the Chinese cabbage crop; (S2) amplifying the target sequence by performing a polymerase chain reaction (PCR) by mixing the template and the primer set described above with the DNA isolated in step (S1); And (S3) separating the PCR product amplified in the step (S2) by size.

In one embodiment of the invention, amplification of the target sequence comprises (i) an initial pre-denaturation process; (Ii) repeating a cycle several times to several tens of a denaturation process, an annealing process for primers, and an elongation process; And (iii) a final heat treatment process or a thermal cycle program adapted to these processes.

As used herein, the term "cycle" refers to the process of making one copy of a target nucleic acid.

In the present invention, the polymerase chain reaction is a reagent for amplification, such as an appropriate amount of DNA, in addition to the primer set consisting of the primer set consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or the base sequence of SEQ ID NO: 3 and SEQ ID NO: 4 of the present invention Polymerases (eg, thermally stable DNA polymerases from Thermus aquatiucs (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis or Phyrococcus furiosis (Pfu)), DNA polymerase cofactors (Mg ²⁺ ), Buffers, dNTPs (dATP, dCTP, dGTP and dTTP) and water (dH ₂ O). The buffer solution may include, but is not limited to, an appropriate amount of Triton X-100, dimethylsulfoxide (DMSO), Tween20, nonidet P40, PEG 6000, formamide and bovine serum albumin (BSA). In addition it can be done using.

In the present invention, the base sequence determination of the amplified target sequence is direct sequencing, DNA microarray, temperature gradient gel electroresis (TGG), single strand conformation polymorphism (SSCP), denaturing high DHPLC It can be explored using various DNA assays known in the art, such as performance liqueid chromatography (HRM), high-resolution melting (HRM) analysis, and preferably, sequencing or high-resolution melting (HRM) analysis. It is good to use

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material emitting fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer, so that the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. One or more sets of oligonucleotide primers used to amplify the target sequence are as described above.

The method includes detecting the amplification product. Detection of the amplification product may be performed through DNA chip, gel electrophoresis, sequencing, radioactivity, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting amplification products, gel electrophoresis can be performed. Gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product.

According to one specific embodiment of the present invention, two DNA fragments of Xcc_47R1 and Xcc_85R1 specific for Xcc race 1 can be identified, such that race-specific primers can be designed using these specific DNA fragments.

According to one specific embodiment of the present invention, the specificity of each primer set after primer design can be confirmed by PCR using genomic DNA (gDNA) isolated from Xcc race and other bacterial strains.

According to one specific embodiment of the present invention, PCR pairs of primers designed from the Xcc race 1 sequence, Xcc_47R1, amplified only the DNA samples of race 1, and primer pairs designed from Xcc_85R1 amplified race 1, although DNA samples of other races were also amplified. Could be distinguished.

When using the primer set according to an embodiment of the present invention, the sequencing method may use a Sanger method, microelectrophoretic sequencing, pyro sequencing, nano-pore single DNA sequencing and the like. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash Radioactivity can be measured using a liquid scintillation counter.

According to one specific embodiment of the invention, two different SCAR markers Xcc_47R1 and Xcc_85R1 can distinguish Xcc race 1 from all other races of Xcc and Xanthomonas strains.

By using the molecular marker according to the present invention to determine the black rot fungus race 1 of the Chinese cabbage and crops can be more simply and accurately determine the black rot of the Chinese cabbage and crops, to reduce the time, cost and effort required for breeding of Chinese cabbage and crops It is expected to be very useful.

1 is a black rot ( Xcc ) race (1, 3, 4, 9), X. campestris pathovars of Chinese cabbage crops ( Xci, Xcr ) and the whole genome sequence alignment of the pepper bacterium spotty bacteria ( X. euvesicatoria ).
Figure 2 shows agarose gel electrophoresis of PCR products using SCAR markers to amplify specific sequences from genomic DNA of Xcc race (race 1-7) and X. campestris pathovar ( Xci, Xcr, Xcz ), Xanthomonas strains and other phytopathogens . It is the result.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Materials and methods

(1) bacterial strains and culture conditions

The bacterial strains used in the present invention are shown in Table 1. Xcc race (race 1-7), Xc pathovar WHRI-6377 ( Xci ) and WHRI-8305 ( Xcr ) were provided by the Resource Center at the Welles Bourne Campus (HRIW) at the University of Warwick. Xanthomonas strains (KACC10490, KACC11153, KACC17821, KACC17126, KACC10491) are available from the Korea Rural Development Administration Agricultural Life Resource Center (KACC), other plant pathogens ICMP13051 and ICMP12464 are Landcare Research, Auckland, New Zealand, and NIHHS1326 Obtained from the Academy of Sciences. Plasmodiophora brassicae (Pathotype1) -Gangneung-1 was retained in the laboratory, and all bacterial strains were incubated for 48 hours at 30 ° C in King's medium B (KB).

(2) extraction of genomic DNA

Genomic DNA of all bacterial strains was extracted using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The concentration and purity of the extracted DNA were measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA).

(3) order search and sort

In silico analysis was performed to design primers to specifically detect strains of Xcc race 1. For comparative analysis, the Xcc strains B100 (race 1), ATCC 33913 (race 3), CFBP5817 (race 4) and Str. 8004 (race 9) and Xanthomonas pathovars ( Xci (CFBP1606R), Xcr (756C)) and X euvesicatoria - Xev (Str . 85-10 ) downloaded the entire genome sequence from NCBI (http://www.ncbi.nlm.nih.gov).

Xcc race and X. The genome size of the campestris pathovars was between 4.91 and 5.17 Mbp, and the G + C content was in the range of 64.2 and 65.3, indicating that there may be large nucleotide variations. Using Mauve (version 2.4.0) and Geneious (free trial version), the sequences of the genomes were aligned, and the blocks showing homology between races of Xcc were marked with the same color and the other blocks were identified with their respective colors. . This method facilitated the development of primers of specific races by identifying differences between strains.

Example 1 Primer Design and PCR Conditions

Primers using the using the software Primer3 (www.frodo.wi.nit.edu) designed from the genome sequence of the bacterial "In silico simulation of molecular biological experiments software" (www.insilico.ehu.es) In silico Specificity was confirmed at. Highly conserved regions were excluded from the primer design. Only variable regions were selected that could provide race-specific sequences needed to identify Xcc race 1. Race-specific primer sets were designed with two sets for race 1. As shown in Table 2 below, the primers Xcc_47R1 and Xcc_85R1 were designed to detect race 1. race 1-specific primers are as follows: Xcc_47R1 designed between genomic positions 498412-4985901 and Xcc_85R1 primers designed between genomic positions 4836126-4836592; These two primers are expected to amplify 1089 and 467 base pairs, respectively.

PCR master mixes (Takara, Shiga, Japan) were used for polymerase chain reaction (PCR) for amplification of target sites. 20.0 μl of PCR reaction mixture containing forward and reverse primers (1.0 μl each), PCR master mix (9.0 μl), ultrapure water (8 μl) and 1.0 μl of DNA was used for PCR amplification. PCR was performed on a gene amplifier (Takara, Shiga, Japan) using the following conditions: denature for 5 minutes at 95 ° C., then denature for 30 seconds at 95 ° C., primer binding for 40 seconds at 65 ° C., at 72 ° C. Elongation for 45 seconds was repeated three times 20 times and ended with a final elongation of 5 minutes at 72 ° C. Electrophoresis was performed for 30 minutes using 1.5% agarose gel at 100 V and visualized in an ENDURO Tm GDS gel documentation system under ultraviolet (302 nm).

Example 2 Confirmation of Specificity of Primer

The specificity of the designed primers was confirmed using BLAST (http: /210.110.86.160/lab/home.html). Laboratory verification Xcc race (race 1-7) and Xc pathovar (Xci, Xcr, Xcz ), Xanthomonas strains (X. campestris, pepper bacterial jeommunuibyeong fungi (X. euvesicatoria), bacterial jeommunuibyeong bacteria (X. axonopodis pv. Dieffenbachiae) beans and fire blight bacteria (X. axonopodis pv. glycines)) and other plant pathogens (bacteria-free black blotch (Pseudomonas syringae pv. maculicola), bacterial soft rot (Erwinia carotovora subsp. carotovora), watermelon and vine crops, blight fungus (Didymella PCR was performed using genomic DNA (1 μl of a suspension of about 60 ng μl ⁻¹ ) of bryoniae ) and Plasmodiophora brassicae . The accuracy and specificity of the primers Xcc_47R1 & Xcc_85R1 developed for race 1 detection were evaluated. Negative controls were evaluated by replacing the DNA template with 1.0 μl DDW.

1 is a black rot ( Xcc ) race (1, 3, 4, 9), Xc pathovars ( Xci, Xcr ) and the whole genome sequence alignment of the pepper bacterium spotty bacteria ( X. euvesicatoria - Xev ). Four Xcc races (race 1, 3, 4, 9) and multiple alignments of the entire genome of Xci, Xcr and Xev consist of several rearranged fragments larger than 1 Kb. Blocks showing sequence homology are shown in the same single color and the same blocks are connected by lines between each genome. The assay bar below shows the location of specific DNA fragments designed for race specific primers in Xcc race 1.

Figure 2 shows agarose gel electrophoresis of PCR products using SCAR markers to amplify specific sequences with genomic DNA of Xcc race (race 1-7) and X. campestris pathovar ( Xci, Xcr, Xcz ), Xanthomonas strains and other phytopathogens . It is the result. The reaction was carried out using primer pairs a) Xcc_47R1_F, Xcc_47R1_R and b) Xcc_85R1_F, Xcc_85R1_R. DNA ladder-100bp; Lanes 1-7: Xcc race 1-7; Lane 8: WHRI-6377 ( Xci ); Lane 9: WHRI-8305 ( Xcr ); Lane 10: Xcz (KACC17126); Lane 11: Xc (KACC10490); Lane 12: Xev (KACC11153); Lane 13: Xad (KACC17821); Lane 14: Xag (KACC10491); Lane 15: Psudomonas syringae pv. maculicola (ICMP13051); Lane 16: Erwinia carotovora subsp. carotovora (ICMP12464); Lane 17: Plasmodiophora brassicae , lane 18: Didymella bryoniae and lane 19: negative control (DDW). DNA concentrations of all samples were 60 ng μl ⁻¹ .

By using the molecular marker according to the present invention to determine the black rot fungus race 1 of the Chinese cabbage and crops can be more simply and accurately determine the black rot of the Chinese cabbage and crops, to reduce the time, cost and effort required for breeding of Chinese cabbage and crops It is expected to be very useful.

<110> Industry-academic cooperation foundation of sunchon national university <120> Molecular marker for selecting Xanthomonas campestris pv.          campestris race 1 of Brassicaceae crops and selection method          using the same molecular marker <130> PA-17-0277 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Xcc_47R1 forward primer <400> 1 cctcctgagt catggcaatg gc 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Xcc_47R1 reverse primer <400> 2 tagcagggga gtgctgcttg c 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Xcc_85R1 forward primer <400> 3 gcggctcggc ttcacggtca gc 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Xcc_85R1 reverse primer <400> 4 gcccaggatg cagcgcagcg t 21

Claims (3)

Primer set for determining the SEQ ID NO: 1 and, baechugwa black rot fungi of crops, including a pair of primers consisting of SEQ ID NO: 2 (Xanthomonas campestris pv. Campestris (Xcc)) first race. (S1) separating the DNA from the black rot strains of the Chinese cabbage crop;
(S2) amplifying a target sequence by performing a polymerase chain reaction (PCR) by mixing the DNA isolated in step (S1) with a template and a primer set according to claim 1; And
(S3) separating the PCR product amplified in step (S2) by size
(Campestris (Xcc) Xanthomonas campestris pv .) Baechugwa black rot fungi of crops including a method of determining the race 1. Black rot bacterium ( Xcc ) race 1 discrimination kit of Chinese cabbage and crops comprising the primer set of claim 1.

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