CN111057777B - Molecular marker and primer pair for identifying xanthomonas campestris 9 physiological race and application thereof - Google Patents
Molecular marker and primer pair for identifying xanthomonas campestris 9 physiological race and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker, a primer pair and application thereof for identifying number 9 physiological microspecies of xanthomonas campestris. The invention firstly discloses a primer pair for identifying or assisting in identifying the number 9 physiological race of xanthomonas campestris, which consists of a primer Xcc9-F and a primer Xcc 9-R: the primer Xcc9-F is a single-stranded DNA molecule shown in SEQ ID NO. 1; the primer Xcc9-R is a single-stranded DNA molecule shown in SEQ ID NO. 2. The invention further discloses a method for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race. The method for identifying the physiological race of the xanthomonas campestris 9 can be effectively used for identifying the physiological race of the xanthomonas campestris 9, and effective prevention and control measures are taken aiming at the physiological race to lay a theoretical foundation for the prevention and treatment of black rot and disease-resistant breeding.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a molecular marker and a primer pair for identifying number 9 physiological microspecies of xanthomonas campestris and application thereof.
Background
Black rot is caused by Xanthomonas campestris (Xcc), a devastating seed-borne disease that infects almost all cruciferous vegetables, including cabbage, broccoli, cauliflower, brussels sprouts, radish, turnip, kohlrabi, etc., affecting yield and commodity, thereby causing huge economic loss. The most significant source of infestation of black rot is from infected seeds and plant residues, while it also survives in cruciferous grasses and ornamentals. In addition, the black rot can be spread by rain, wind, irrigation and agricultural equipment. Under natural conditions, pathogenic bacteria invade the host mainly from wounds, leaf water pores and insects, then propagate and spread along leaf vascular tissues, then blacken veins, withered yellow and necrotic mesophyll tissues and finally form V-shaped disease spots typical of black rot along the veins. The disease can be attacked in the seedling stage and the adult stage of the cabbage, and mainly affects leaves; after the black rot disease occurs, the commodity of the cabbages is seriously influenced, the yield of the cabbages can be reduced by 50 percent, even the failure of harvest can be caused under the proper climatic condition, and thus huge economic loss is caused.
Black rot was first discovered in the united states as early as the 90 s of the 19 th century, and later developed in a wide range worldwide. The xanthomonas campestris has diverse physiological species differentiation, and 11 physiological species exist, so the control of the xanthomonas campestris is also quite difficult. The pathogenic response caused by different race types varies, and thus the prerequisite for the prevention and control of the disease is the identification of the prevalent race type. The physiological race analysis is carried out on the xanthomonas campestris germs, so that the information of the composition, distribution frequency, change between years and the like of the germ races can be known, and a reference is provided for controlling the disease prevalence in the layout of disease-resistant varieties. The conventional microspecies identification method is a host identification method, is systematic, but is time-consuming and labor-consuming, is not suitable for quickly identifying a large number of pathogenic bacteria, has a relatively long period, and is easily influenced by inoculation conditions and environmental conditions to cause poor reliability.
Therefore, a simple and effective molecular marker technology is developed to identify the xanthomonas campestris physiological race, a new way is provided for the identification of the xanthomonas campestris physiological race, and the method is a prerequisite condition for breeding the black rot resistance.
Disclosure of Invention
The invention aims to solve the technical problem of how to quickly and accurately identify the number 9 physiological race of the xanthomonas campestris.
In order to solve the technical problems, the invention firstly provides a primer pair for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race.
The primer pair for identifying or assisting in identifying the number 9 physiological race of the xanthomonas campestris consists of a primer Xcc9-F and a primer Xcc 9-R:
the primer Xcc9-F is a single-stranded DNA molecule shown in SEQ ID NO. 1; the primer Xcc9-R is a single-stranded DNA molecule shown in SEQ ID NO. 2.
The kit containing the primer pair is also within the protection scope of the invention, and the kit is a kit for identifying or assisting in identifying the number 9 physiological race of xanthomonas campestris.
The preparation method of the kit comprises the step of packaging each primer of the primer pair separately.
The invention further provides an application of the primer pair or the kit in any one of the following steps:
A1) preparing a product for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race;
A2) identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race;
A3) preparing a product for identifying or assisting in identifying whether the Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race or not;
A4) identifying or assisting in identifying whether the xanthomonas campestris to be detected is xanthomonas campestris No. 9 physiological race or not;
A5) preparing a product for detecting or assisting in detecting the xanthomonas campestris No. 9 physiological race;
A6) detecting or detecting the xanthomonas campestris No. 9 physiological race in an auxiliary way;
A7) preparing a product for detecting or assisting in detecting whether a sample to be detected contains xanthomonas campestris No. 9 physiological race or not;
A8) detecting or detecting in an auxiliary way whether the sample to be detected contains xanthomonas campestris No. 9 physiological race.
The invention further provides a method for detecting or assisting in identifying whether the xanthomonas campestris to be detected is xanthomonas campestris # 9 physiological race.
The invention discloses a method for detecting or assisting in identifying whether Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race, which comprises the following steps:
taking the genome DNA of the Xanthomonas campestris to be detected as a template, and carrying out PCR amplification by adopting the primer pair or the kit pair to obtain a PCR amplification product;
if the PCR amplification product contains a strip with the size of 1000-1200bp, the Xanthomonas campestris to be detected is or is selected as Xanthomonas campestris No. 9 physiological race; otherwise, the to-be-detected xanthomonas campestris is not or is not a candidate of xanthomonas campestris No. 9 physiological race.
The invention discloses a method for detecting or assisting in detecting whether a sample to be detected contains xanthomonas campestris No. 9 physiological races or not, which comprises the following steps:
taking the genome DNA of a sample to be detected as a template, and carrying out PCR amplification by adopting the primer pair or the kit pair to obtain a PCR amplification product;
if the PCR amplification product contains a strip with the size of 1000-1200bp, the sample to be detected contains or is candidate to contain the xanthomonas campestris No. 9 physiological race; otherwise, the sample to be detected does not contain or is candidate to contain the xanthomonas campestris No. 9 physiological race.
In the above method, the 1000-1200bp band may be a 1092bp band.
The reaction conditions for the PCR amplification of the present invention are as follows: pre-denaturation at 94 deg.C for 5 min; 38 cycles of 94 ℃ for 30s, 60 ℃ for 1min and 72 ℃ for 30 s; extension for 10min at 72 ℃.
The invention further provides a molecular marker for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race.
The molecular markers of the invention are C1) or C2) as follows:
C1) a DNA molecule of SEQ ID No. 3;
C2) DNA molecules having a homology of more than 90% with the DNA molecule of SEQ ID NO. 3.
The 90% or greater homology may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% homology.
The application of the molecular marker in any one of the following is also within the protection scope of the invention:
B1) preparing a product for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race;
B2) identifying or assisting in identifying xanthomonas campestris No. 9 physiological race;
B3) preparing a product for identifying or assisting in identifying whether the Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race or not;
B4) identifying or assisting in identifying whether the xanthomonas campestris to be detected is xanthomonas campestris No. 9 physiological race or not;
B5) preparing a product for detecting or assisting in detecting the xanthomonas campestris No. 9 physiological race;
B6) detecting or detecting the xanthomonas campestris No. 9 physiological race in an auxiliary way;
B7) preparing a product for detecting or assisting in detecting whether a sample to be detected contains xanthomonas campestris No. 9 physiological race or not;
B8) detecting or detecting in an auxiliary way whether the sample to be detected contains xanthomonas campestris No. 9 physiological race.
The invention also provides a method for identifying or assisting in identifying whether the Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race.
The invention discloses a method for identifying or assisting in identifying whether Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race, which comprises the following steps:
detecting whether the molecular marker exists in the genome DNA of the Xanthomonas campestris to be detected, and if the molecular marker exists, determining that the Xanthomonas campestris to be detected is or is selected as a No. 9 physiological race of the Xanthomonas campestris; if the molecular marker does not exist, the Xanthomonas campestris to be detected is not or is not a candidate of Xanthomonas campestris No. 9 physiological race.
The invention also provides a method for detecting or assisting in detecting whether the sample to be detected contains xanthomonas campestris 9 physiological race.
The method for detecting or assisting in detecting whether the sample to be detected contains xanthomonas campestris No. 9 physiological race or not comprises the following steps:
detecting whether the molecular marker exists in the genome DNA of a sample to be detected, if so, determining that the sample to be detected contains or is candidate to contain xanthomonas campestris No. 9 physiological race; if the molecular marker does not exist, the sample to be detected does not contain or is candidate to contain the xanthomonas campestris No. 9 physiological race.
In the above, the sample to be tested is any one of the following:
E1) a monocot plant;
E2) a dicotyledonous plant;
E3) a cruciferous plant;
E4) cabbage.
The molecular marker is the first indel marker linked with the xanthomonas campestris No. 9 physiological race at present, and the marker and a primer pair designed aiming at the marker can be effectively used for identifying the xanthomonas campestris No. 9 physiological race; when the cruciferous crops suffer from black rot, DNA of the diseased leaves can be extracted to quickly identify whether the cruciferous crops are the No. 9 physiological race or not, so that effective prevention and control measures are taken for the physiological race, and a theoretical basis is laid for prevention and control of the cruciferous crops suffering from black rot and disease-resistant breeding.
Drawings
FIG. 1 is an electrophoresis diagram of PCR products using genomic DNAs of different physiological races as templates.
FIG. 2 is an electrophoresis diagram of PCR products using genomic DNA of No. 9 race at different concentrations as templates, wherein 1, 5, 10, 25, 50, 100, and 150 represent genomic DNA of No. 9 race at concentrations of 1 ng/. mu.L, 5 ng/. mu.L, 10 ng/. mu.L, 25 ng/. mu.L, 50 ng/. mu.L, 100 ng/. mu.L, and 150 ng/. mu.L, respectively.
FIG. 3 is the electrophoresis picture of PCR products using genome DNA of diseased leaves inoculated with different physiological races as templates.
FIG. 4 is the electrophoresis diagram of PCR products using genomic DNA of diseased leaves inoculated with physiological race 9 at different concentrations as templates, wherein 1, 5, 10, 25, 50, 100, 150 represent genomic DNA of diseased leaves at concentrations of 1 ng/. mu.L, 5 ng/. mu.L, 10 ng/. mu.L, 25 ng/. mu.L, 50 ng/. mu.L, 100 ng/. mu.L and 150 ng/. mu.L, respectively.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, Xanthomonas campestris No.1 (HRIW-3811), Xanthomonas campestris No.2 (HRIW-3849A), Xanthomonas campestris No.3 (HRIW-5212), Xanthomonas campestris No. 4 (HRIW-1279A), Xanthomonas campestris No. 5 (HRIW-3880), Xanthomonas campestris No. 6 (HRIW-6181), Xanthomonas campestris No. 7 (HRIW-8450A), Xanthomonas campestris No. 9 (Xcc8004) are described in non-patent documents "Khand S A et al.development of Molecular Marker Genome reaction for specificity Detection of Xanthomonas campestris pv. campestis Race 5, a Pathologen of Black roll disease. J.Microbiol. Bioh. Biotech.2019), and university, Microbiol.795, A.795, university, Kogaku gaku ga, the biological material is only used for repeating the related experiments of the invention, and can not be used for other purposes.
The inventor finds the unique indel marker of the xanthomonas campestris No. 9 physiological race, and the marker is a DNA molecule shown in SEQ ID NO. 3.
Example 1 identification of Xanthomonas campestris No. 9 physiological races by Using bacterial genomic DNA
Design of indel primer
The primer sequence is as follows:
Xcc9-F：5’-CATGGCAGATGTCTCCGTGA-3’(SEQ ID NO.1)；
Xcc9-R：5’-TTTGCCATCGGCACCAAGCT-3’(SEQ ID NO.2)。
secondly, detecting 8 standard strains by using indel primers
1. Genomic DNAs of a standard strain Xanthomonas campestris No.1 physiological race (HRIW-3811), a standard strain Xanthomonas campestris No.2 physiological race (HRIW-3849A), a standard strain 3 physiological race (HRIW-5212), a standard strain 4 physiological race (HRIW-1279A), a standard strain 5 physiological race (HRIW-3880), a standard strain 6 physiological race (HRIW-6181), a standard strain 7 physiological race (HRIW-8450A) and a standard strain 9 physiological race (Xcc8004) are respectively extracted.
The specific experimental steps are as follows:
streaking the preserved strains on an LB (Langmuir-Blodgett) plate, selecting positive clones after 24 hours, and placing the positive clones in 5mL of LB liquid to shake and shake the colonies for 24 hours;
taking the bacteria culture solution to a 1.5mL centrifuge tube, centrifuging at 10000rpm for 1min, discarding the supernatant, and repeating twice;
adding 200 mu L of buffer solution GA into the thallus precipitate, and oscillating until the thallus is completely suspended;
adding 20 mu L of protease K solution into the centrifuge tube, and turning upside down and mixing uniformly;
adding 220 μ L buffer GB, shaking for 15sec, standing at 70 deg.C for 10 min;
adding 220 μ L of anhydrous ethanol, and fully shaking and mixing for 15 sec;
adding all solution and flocculent precipitate in the centrifuge tube into adsorption column CB3 (placing the adsorption column into the collection tube), centrifuging at 12000rpm for 30sec, pouring off waste liquid, and placing adsorption column CB3 into the collection tube;
adding 500 μ L buffer GD into adsorption column CB3, centrifuging at 12000rpm for 30sec, discarding waste liquid, and placing adsorption column CB3 into a collection tube;
adding 600 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000rpm for 30sec, discarding the waste liquid, and placing adsorption column CB3 into a collection tube;
the column was centrifuged at 12000rpm for 2min and the waste liquid was decanted. Placing the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
transferring the adsorption column CB3 into a new centrifuge tube, suspending 50 μ L of elution buffer TE into the middle part of the adsorption membrane, placing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.
The Xanthomonas campestris genome DNA extracted in the above steps can be directly used for PCR reaction.
2. And (2) respectively taking the genomic DNA of the xanthomonas campestris No.1 physiological race, the physiological race No.2, the physiological race No.3, the physiological race No. 4, the physiological race No. 5, the physiological race No. 6, the physiological race No. 7 and the physiological race No. 9 obtained in the step 1 as a template, and carrying out PCR amplification by using a primer pair (Xcc9-F and Xcc9-R) consisting of Xcc9-F and Xcc9-R to obtain a PCR amplification product.
The PCR reaction system is as follows:
and (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5 min; 38 cycles of 94 ℃ for 30s, 60 ℃ for 1min and 72 ℃ for 30 s; extension for 10min at 72 ℃.
3. And (3) carrying out 2% agarose gel electrophoresis on the PCR amplification product, staining the product by ethidium bromide, and observing the product under an ultraviolet lamp. As shown in FIG. 1, only the genomic DNA of Xanthomonas campestris No. 9 race amplified a 1092bp band, and none of the remaining races showed a 1092bp band. The results show that the primer pair consisting of Xcc9-F and Xcc9-R is adopted to identify the xanthomonas campestris No. 9 physiological race from the bacterial genome DNA.
Example 2 detection sensitivity of the molecular marker Using No. 9 physiological race genomic DNA
Genomic DNA of Xanthomonas campestris No. 9 race (Xcc8004) was extracted according to step 1 of example 1, and diluted to concentrations of 1ng/μ L, 5ng/μ L, 10ng/μ L, 25ng/μ L, 50ng/μ L, 100ng/μ L and 150ng/μ L using genomic DNA of Xanthomonas campestris No. 9 as a template, PCR amplification was performed using a primer pair consisting of Xcc9-F and Xcc9-R (Xcc9-F and Xcc9-R) (PCR reaction system and PCR reaction conditions were as in example 1) to obtain PCR products, and sensitivity of the primer pair Xcc9-F and Xcc9-R to genomic DNA of Xanthomonas campestris No. 9 was examined.
And (3) carrying out 2% agarose gel electrophoresis on the PCR amplification product, staining the product by ethidium bromide, and observing the product under an ultraviolet lamp. As shown in FIG. 2, 1092bp bands were amplified from the bacterial genomic DNA templates at all concentrations, except that 1092bp bands were not amplified from the bacterial genomic DNA templates at 1 ng/. mu.L. The results show that the primer pair consisting of the Xcc9-F and the Xcc9-R can identify the xanthomonas campestris No. 9 physiological race from the bacterial genome DNA template with the concentration of 5 ng/. mu.L.
Example 3 identification of Xanthomonas campestris No. 9 physiological races by diseased plant leaf DNA
1. Cabbage leaves are respectively inoculated with No.1 physiological race (HRIW-3811), No.2 physiological race (HRIW-3849A), No.3 physiological race (HRIW-5212), No. 4 physiological race (HRIW-1279A), No. 5 physiological race (HRIW-3880), No. 6 physiological race (HRIW-6181), No. 7 physiological race (HRIW-8450A) and No. 9 physiological race (Xcc8004), and the specific steps are as follows:
1) taking out the test tube bacteria from the refrigerator, picking a small amount of bacteria from the test tube by the sterilized inoculating loop on a clean bench, and streaking and inoculating the bacteria to an LB plate culture medium. And then culturing the inoculated flat plate in a thermostat at 28 ℃ for 24-48 h, and growing bacterial colonies for later use.
2) Adding a small amount of sterile water into a flat plate with grown bacterial colonies, slightly vibrating to disperse the bacterial colonies in water, taking 2mL of the bacterial colonies in a graduated test tube, and adding sterile water to dilute the bacterial liquid to OD600Tween 20(1ml/L) was added to enhance the adsorption capacity of the bacterial liquid (0.1).
3) And (3) moisturizing 4-6 (about four weeks) true leaf seedlings for 12-24 hours before inoculation, uniformly spraying the bacterial suspension on the cabbage seedlings by using a medical throat sprayer until liquid drops flow down, moisturizing the inoculated seedlings for 12-24 hours, observing the disease occurrence condition after two weeks, and shearing disease occurrence leaves.
2. Shearing the diseased leaves of No.1 physiological races, No.2 physiological races, No.3 physiological races, No. 4 physiological races, No. 5 physiological races, No. 6 physiological races, No. 7 physiological races and No. 9 physiological races, and extracting the genomic DNA of the diseased leaves; the method comprises the following specific steps:
extraction of diseased leaf DNA using CTAB: opening the water bath kettle, and preheating at 65 ℃; taking the leaves of the diseased part, shearing the leaves into 1 multiplied by 1cm, adding 5 steel balls with 2mm, adding 400 microliter 2 multiplied by CTAB, crushing by a tissue crusher, and then adding 200 microliter 2 multiplied by CTAB; water bath at 65 deg.C for 1 hr, shaking for 2-3 times to ensure full cracking, taking out, and cooling to room temperature; adding 500 μ l chloroform/isoamyl alcohol (24: 1), turning upside down, mixing, standing for 5min, and centrifuging at 12000rpm for 10 min; sucking the supernatant (about 400 microliters) to a new sterile centrifuge tube of 1.5mL, adding isopropanol of 2/3 volumes, turning upside down and mixing uniformly, and standing at 4 ℃ for more than 30 min; centrifuging at 12000rpm for 10min, and discarding the supernatant; adding 500 microliter of 75% alcohol, and centrifuging at 12000rpm for 5 min; the supernatant was discarded to obtain plant DNA, and 50TE buffer was added for further use.
3. The genome DNA of the diseased leaves of the No.1 physiological race, the No.2 physiological race, the No.3 physiological race, the No. 4 physiological race, the No. 5 physiological race, the No. 6 physiological race, the No. 7 physiological race and the No. 9 physiological race is taken as a template, and a primer pair consisting of Xcc9-F and Xcc9-R is used for PCR amplification to obtain a PCR amplification product.
The PCR reaction system is as follows:
and (3) PCR reaction conditions: performing pre-denaturation at 94 deg.C for 5 min; 38 cycles of 94 ℃ for 30s, 60 ℃ for 1min and 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
4. And (3) carrying out 2% agarose gel electrophoresis on the PCR amplification product, staining the product by ethidium bromide, and observing the product under an ultraviolet lamp.
As shown in FIG. 3, only the genomic DNA of the diseased leaf of the physiological race inoculated with Xanthomonas campestris No. 9 amplified a 1092bp band, and the DNA of the diseased leaf of the other physiological race did not show a 1092bp band. The result shows that the primer pair consisting of the Xcc9-F and the Xcc9-R is adopted to identify the xanthomonas campestris No. 9 physiological race from the genome DNA of the diseased plant.
Example 4 detection of sensitivity of the molecular marker Using No. 9 physiological race pathogenic leaf DNA
Genomic DNA of diseased leaves inoculated with Xanthomonas campestris No. 9 physiological race (Xcc8004) to cabbage leaves in example 3 was obtained as a template, diluted to concentrations of 1 ng/. mu.L, 5 ng/. mu.L, 10 ng/. mu.L, 25 ng/. mu.L, 50 ng/. mu.L, 100 ng/. mu.L and 150 ng/. mu.L, PCR-amplified with a primer pair consisting of Xcc9-F and Xcc9-R (PCR reaction system and PCR reaction conditions were as in example 3), and sensitivity of the primer pair Xcc9-F and Xcc9-R to genomic DNA of diseased leaves was examined.
And (3) carrying out 2% agarose gel electrophoresis on the PCR amplification product, staining the product by ethidium bromide, and observing the product under an ultraviolet lamp. As shown in FIG. 4, 1092bp bands were amplified from all the leaf DNAs except for the diseased leaf DNA template at a concentration of 1 ng/. mu.L and 5 ng/. mu.L. The result shows that the primer pair consisting of the Xcc9-F and the Xcc9-R can identify the xanthomonas campestris No. 9 physiological race from the leaf genome DNA template with the concentration of 25 ng/. mu.L.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> agriculture and forestry academy of sciences of Beijing, Jing research and Yinong (Beijing) seed industry science and technology Co., Ltd
<120> molecular marker and primer pair for identifying xanthomonas campestris No. 9 physiological race and application thereof
<130> GNCFY192659
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catggcagat gtctccgtga 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tttgccatcg gcaccaagct 20
<210> 3
<211> 1092
<212> DNA
<213> Xanthomonas campestris (Xanthomonas campestris pv. campestris)
<400> 3
catggcagat gtctccgtga cggttgccga ggctggggtg gccgccgtgt gggcgcatgc 60
gcccagagcg aggctggcga gcaaggcgta gagggggcgc gcgatcatga agcgtccgtt 120
gtgcgctggt gtggctgcga cgctagtggc atcgggcgct gaggtcaatc gcaaggcttc 180
tggcagtgat cgacgtgaca cttggtaact ccaggctcat tgccgaaatc attcaaaact 240
ttcaaagcat caggactgag tgacatgtcc gctccttgga cttaacggat gtagacccat 300
tcgacgcatt tgtgcgctac caacgcttgc ggttccgcag cctcaccacg cggaaaaacc 360
tctactcgca ttcctgggcg cgtaaagaaa tcactcatcc agcgcccgtc ttcgacgatg 420
ttgcgctcac gcgcaaatcc catctgctca agctgctggg taaaacgatc aaagtccagg 480
cgacaaagtt cggtcatcgc aggcgatgca ccggccacat tgcgatcgaa gcggaattcg 540
aaccatctgc catcaagctt ggtttcatcg actccaaatc cataactcca atccttagtg 600
agcgggccga aggcccggta tcggcgttcg tcgcctttca cgaactgaac cgcctgcccc 660
atcgtggaat tcagacgctc gggagtgaag tcggagatgt tctggctgcc cttgatcaac 720
acaagcagct gaccaagcaa gccttccgca gtaggcttgt caggcggcac agtggcaggg 780
ctggatgaac tcagagcgga tgtctccgtg acggttgccg gagctgcgcc gcccgccgcg 840
tgggcgcaag ctcccagagc cagggtggcg agcaaggcgt agagaggtcg cgcgatcatg 900
aagcgtcctt agtgcgctga tgtcgctgcg acgctagtgg catgcggcac tgaggtcaat 960
cgcaggtttc tgtcacccgc accatcatcg ccgcacgcta ctcgccctgg ccggcgcctc 1020
cgccggcggc aacacctcca tcagcagctg ccccgcgctg gcggtgatgc gcagcttggt 1080
Claims (5)
1. The primer pair for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race is characterized in that: the primer pair consists of a primer Xcc9-F and a primer Xcc 9-R:
the primer Xcc9-F is a single-stranded DNA molecule shown in SEQ ID NO. 1; the primer Xcc9-R is a single-stranded DNA molecule shown in SEQ ID NO. 2.
2. The kit for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race is characterized in that: the kit comprises the primer pair of claim 1.
3. Use of the primer pair of claim 1 or the kit of claim 2 in any one of:
A1) preparing a product for identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race;
A2) and identifying or assisting in identifying the xanthomonas campestris No. 9 physiological race.
4. The method for detecting or assisting in identifying whether the xanthomonas campestris to be detected is xanthomonas campestris 9 physiological race or not is characterized by comprising the following steps of: the method comprises the following steps:
taking genome DNA of Xanthomonas campestris to be detected as a template, and carrying out PCR amplification by adopting the primer pair of claim 1 or the kit of claim 2 to obtain a PCR amplification product;
if the PCR amplification product is a single band with the size of 1092bp, the Xanthomonas campestris to be detected is or is selected as Xanthomonas campestris No. 9 physiological race; otherwise, the to-be-detected xanthomonas campestris is not or is not a candidate of xanthomonas campestris No. 9 physiological race.
5. The method for identifying or assisting in identifying whether the Xanthomonas campestris to be detected is Xanthomonas campestris No. 9 physiological race or not is characterized by comprising the following steps: the method comprises the following steps:
detecting whether a molecular marker exists in the genome DNA of the xanthomonas campestris to be detected, wherein if the molecular marker exists, the xanthomonas campestris to be detected is or is selected as a physiological race of xanthomonas campestris No. 9; if the molecular marker does not exist, the Xanthomonas campestris to be detected is not or is not a candidate of Xanthomonas campestris No. 9 physiological race;
the molecular marker is a DNA molecule shown as SEQ ID NO.3 in a sequence table.
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| KR20120053831A (en) * | 2010-11-18 | 2012-05-29 | 대한민국(농촌진흥청장) | Primer set of polymerase chain reaction for detecting xanthomonas campestris pv. incanae and method for detecting xanthomonas campestris pv. incanae using the same |
| KR20150074845A (en) * | 2013-12-24 | 2015-07-02 | 대한민국(농촌진흥청장) | Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae |
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| US5420009A (en) * | 1992-03-23 | 1995-05-30 | The United States Of America As Represented By The Secretary Of Agriculture | Detection of Xanthomonas campestris pv. citri by hybridization and polymerase chain reaction assays |
| KR100514431B1 (en) * | 2003-03-06 | 2005-09-14 | 대한민국 | Primer Sets for Specifid Identification of a Pathogen Xanthomonas campestris pv. campestris, and Specific-Identification Methods of the Pathogen using the Primers |
| CN102187874B (en) * | 2010-03-16 | 2013-04-24 | 广西大学 | Application of pathogenic gene related to xanthomonas campestris pathovar campestris |
| CN102108407B (en) * | 2010-12-20 | 2012-10-24 | 北京市农林科学院 | Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof |
| CN102373199B (en) * | 2011-11-08 | 2012-11-21 | 北京市农林科学院 | Reagent kit for identifying xanthomonas campestris pv. campestris No.4 physiological race |
| CN103114136A (en) * | 2013-01-25 | 2013-05-22 | 郑媛 | Fluorescent PCR (polymerase chain reaction) quick detection primer and kit for Xanthomonas campestris pv. campestris |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20120053831A (en) * | 2010-11-18 | 2012-05-29 | 대한민국(농촌진흥청장) | Primer set of polymerase chain reaction for detecting xanthomonas campestris pv. incanae and method for detecting xanthomonas campestris pv. incanae using the same |
| KR20150074845A (en) * | 2013-12-24 | 2015-07-02 | 대한민국(농촌진흥청장) | Method for making prove or primer for detecting Xanthomonas campestris pv. lespedezae |
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