Molecular markers of xanthomonas oryzae pv oryzae and application thereof

A molecular marker and rice technology, applied in the field of plant genetic engineering, can solve problems such as poor identification accuracy and unreliable identification technology, and achieve the effect of improving detection reliability

Inactive Publication Date: 2015-11-18
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Finally, the molecular markers that can be used to distinguish are determined as follows: the molecular marker of rice bacterial blight, whose sequence is shown in SEQ ID NO.1-58; the molecular marker of rice bacterial streak bacterium, whose sequence is shown in SEQ ID NO.59-104 Shown; The co-dominant molecular markers of Xanthomonas oryzae and Bacterial spot of rice, its sequence is shown in SEQIDNO.105-126; Using these molecular markers can not only specifically identify Xanthomonas oryzae, but also can The two pathogenic varieties of Xanthomonas oryzae Xanthomonas oryzae Xanthomonas oryzae Xanthomonas oryzae and Bacterial leaf spot were further identified in detail, so as to solve the problem of poor identification accuracy and unreliable identification technology in the existing technology

Method used

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  • Molecular markers of xanthomonas oryzae pv oryzae and application thereof
  • Molecular markers of xanthomonas oryzae pv oryzae and application thereof
  • Molecular markers of xanthomonas oryzae pv oryzae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Design of molecular markers that can distinguish and identify rice bacterial blight and rice bacterial streak

[0024] Three races KACC10331, PXO99A, MAFF311018 of Xanthomonasoryza epv.oryzae and two races BLS256 and RS105 of Xanthomonasoryzaepv.oryzicola, the genome sequences and related information of the five pathogens Downloaded from NCBI (http: / / www.ncbi.nlm.nih.gov / ). (Table 1)

[0025] Table 1 Statistics of five bacterial genome information

[0026]

[0027] The inventors compared the whole genomes of three races of Xoo (MAFF311018, KACC10311, PXO99A) and found that the sequence similarity was high, so MAFF311018 was selected as the model race of Xoo.

[0028]Compared with RS105 and BLS256, it was found that BLS256 contained all the information of RS105, with a high degree of similarity, so BLS256 was used as the model race of S. oryzae.

[0029] BLAST was used to compare the whole genome sequences of the two model races MAFF311018 and BLS256, and...

Embodiment 2

[0040] Example 2 Screening and verification of dominant molecular markers for rice bacterial blight specialization

[0041] According to the ePCR screening results of the designed specific primers for Xanthomonas oryzae, we selected 40 pairs of primers to verify against 25 strains of Xanthomonas oryzae and 10 strains of Bacterial blight of rice and other related strains .

[0042] Streak the strains to be tested on the TSA solid medium plate, culture at 28°C for 2 days, pick a single colony, place it in TSA liquid medium, and heat it at 180r min -1 , 28 ° C shaker culture for two days. Aspirate 3mL of the bacterial solution, and use the kit to extract the genomic DNA of the strain. And use it as a template for PCR reaction.

[0043] The optimized reaction system is: Taq enzyme 0.2 μL (5u·μL -1 ), 10×PCRBuffer (Mg 2+ Free) 2μL, 25mM MgCl 2 1.2 μL, 1.5 μL of 2.5mMdNTPMixture, 1 μL of bacterial solution as a template, 0.6 μL of primer F (10 μM), 0.6 μL of primer R (10 μM), ...

Embodiment 3

[0048] Example 3 Screening and Validation of Dominant Molecular Markers Specific to Rice Bacterial Leaf Spot

[0049] According to the ePCR screening results of the specific primers specialized for rice bacterial leaf spot bacterium, adopt the genomic DNA of the test bacterial strain that has obtained, utilize the optimized PCR reaction system identical with embodiment 2 and 2% TBE agarose For the detection method of gel electrophoresis, 40 pairs of primers were selected to verify the tested strains.

[0050] After testing, we obtained 23 pairs of specific primers that can accurately amplify 10 strains of Bacteria sativa in all tested strains to a DNA fragment that matches the target size (see Table 6 for the fragment size). None of these specific primers could amplify bands in Xanthobacterium oryzae and other reference strains (such as image 3 shown).

[0051] Table 6 The sequence numbers of such molecular markers in the sequence listing and the names of the corresponding ...

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Abstract

The invention relates to the technical fields of plant aetiology and plant quarantine and provides a batch of molecular markers capable of conveniently, rapidly and specifically identifying Xanthomonas oryzae, especially dominant molecular markers and codominant molecular markers for identification of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola and application of the molecular markers for detection of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola. With an identification method using each or a mixture of the molecular markers, reliability of results is better ensured, diseases can be accurately identified, which enables correct prevention and treatment measures to be taken and accords with the current development direction in inspection and quarantine, and expansion and spreading of diseases can be timely controlled.

Description

[0001] This application is a divisional application for the invention patent filed by the applicant on April 25, 2014, entitled "Molecular markers of Xanthomonas oryzae and its application" (original application number: 2014101695173). technical field [0002] The invention relates to the technical field of plant genetic engineering, and provides molecular markers and applications of Xanthomonas oryzae, in particular, dominant molecular markers and codominant molecules for identification of rice bacterial blight and rice bacterial streak markers, and the application of these molecular markers to detect two pathogenic bacteria. Background technique [0003] Xanthomonas oryzae is a typical yellow Gram-negative bacteria, which is the main bacterial pathogenic bacteria on rice, including Xanthomonasoryza epv.oryzae (Xoo) and Xanthomonasoryzaepv. oryzicola (Xoc) (Zhao, Ardales et al. 2004). The vascular disease that Xoo can cause in rice is bacterial blight of rice, while Xoc inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
CPCC12Q1/689C12Q2600/156
Inventor 丁新华储昭辉冯雯杰杨龙冯传顺
Owner SHANDONG AGRICULTURAL UNIVERSITY
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