
Bacterial blight is a major disease of ornamental crops, such as stock (
Comparative genomic approach represents direct comparison of the collective genomes of a particular group of organisms either across the genus or species level. It reveals the similarities, differences and evolutionary patterns among the diverse organisms at genomic level (Abby and Daubin 2007; Sivashankari and Shanmughavel 2007). This approach was successful in understanding the evolutionary mechanism of
To date, no molecular marker has been reported to detect
A total of 18 diverse bacterial strains was collected from different sources and used in this study (Supplementary Table S1). Bacterial strains were grown on King’s B media at 30°C for 48 hours. All bacterial strains were stored at –80°C in a liquid medium with 50% glycerol for further uses.
Bacterial DNA was extracted from cultured bacterial cells using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instruction. The quality of isolated DNA was checked using 1.2% agarose gel electrophoresis at 100 V and quantified using a NanoDropND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA).
Comparative genome analysis was done by using EDGAR 3.0 (Blom
Table 1 .
Strain | Xcc B100 | Xcc ATCC33913 | Xcc CFBP5817 | Xcc 8004 | Xcc CN17 | Xcr 756C | Xci CFBP1606R | Xci CFBP2527R | Xev LMG930 | Xcg CFBP2526 | Xad LMG695 | Xac Xac 29 |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Size (Mbp) | 5.07 | 5.07 | 4.97 | 5.14 | 4.99 | 4.94 | 4.96 | 4.92 | 5.07 | 5.363 | 4.99 | 4.60 |
Gene | 4,542 | 4,240 | 4,447 | 4,584 | 4,398 | 4,372 | 4,423 | 4,380 | 4,977 | 4,856 | 4,592 | 4,641 |
Protein | 4,236 | 4,179 | 4,122 | 4,297 | 4,086 | 4,055 | 4,071 | 4,032 | 4,505 | 4,461 | 3,919 | 4,237 |
NCBI ID | NC_010688 | NC_003902 | NZ_CM002673 | NC_007086 | CP011946.1 | NC_017271 | NZ_CM002635 | NZ_CM002636 | NZ_CP018467 | NZ_CM002268 | NZ_CP014347 | NC_020800 |
The EDGAR tool produced information on core genome, dispensable genome and singletons; which were considered for marker development. In addition, genome plot and the gene cluster (Fig. 1) representation were also obtained from the EDGAR tool.
Table 2 . List of primer sequences with expected product size and annealing temperatures for specific amplification of
Primers | Forward (5’-3’) | Reverse (5’-3’) | Expected size (bp) | Annealing temperature (°C) |
---|---|---|---|---|
XCI_1F/R | ATGTCGATCCAGCTCAGGTG | TCAGGTTGCAGGAGGGCTTGG | 495 | 64 |
XCI_2F/R | CCTTCAGGTCATGCACCATTG | ATCCGCATTCTGCCCTCCCCCCT | 503 | 65 |
XCI_3F/R | GCGATGGATACAGTGACTGGAAG | CAACTGCAAGGCCGTATCGTA | 612 | 62 |
XCI_4F/R | CAGGCGGCCGATGCGAAGG | TGATGTTGAAGAGTGTGCGGAC | 604 | 63 |
XCI_5F/R | CATTCTCGCGGAGCGGCGTCTGC | AGCTGCTGAGTTGCCGCGCTCT | 665 | 68 |
XCI_6F/R | TCTGGCTCCCACTATGTGC | CGGGATGGATGAGGCTGTCC | 468 | 60 |
XCI_7F/R | GTTCGGACTGGCGCTCTGGC | TCTTCATCATCACTAACCCA | 303 | 60 |
XCI_8F/R | GATCATGGTGTCGCCCATG | TTAAGCCACTTTGAGCTGCACC | 324 | 60 |
XCI_9F/R | GTGACCCTGCGATACATCGG | GGTGCTGCCAAGGCCGGAGC | 769 | 65 |
XCI_10F/R | GGTACTACTGGATGCCAACC | TCAGGCCTTCATAAGATTTTG | 397 | 57 |
XCI_11F/R | ATGGATGCCGCTGCCGGTCG | GCGGGTAGGTGAGGCCGTCGA | 595 | 65 |
Specificity of the designed primer was determined by the PCR assay using extracted DNA from strain of each of the
The bacterial DNA sample was diluted sequentially in ten fold increments using sterile double distilled water up to a dilution of 10-9 and fixed the DNA concentration ranged from 7 ng/µL to 0.00000007 ng/µL for detection of the sensitivity level of the well amplified
Comparative genomic approach was implied to identify the genetic similarities and variations among the diverse
(Fig. 2). Comparative genome analysis conferred the segregation of the genomes into three major categories: 1) core genome, 2) dispensable genome and 3) singletons. The core genome consisted of 70% of the total gene (2,834) which are conserved across the genome of the bacterial strains, dispensable or flexible genome contained 1,108 genes which covered about 28% of the total number of genes and the remaining 2% comprised 82 singleton genes, which were found only in
Table 3 . Molecular marker information for specific detection of
Primers | Gene name | Start | End | Length (bp) | Direction | Protein description |
---|---|---|---|---|---|---|
XCI_1F/R | 3641561 | 3641067 | 495 | - | Hypothetical protein | |
XCI_2F/R | 2955263 | 2956369 | 1,107 | + | Hypothetical protein | |
XCI_3F/R | 4861522 | 4862766 | 1,245 | + | Hypothetical protein | |
XCI_5F/R | 886319 | 884361 | 1,959 | - | Hypothetical protein | |
XCI_6F/R | 196812 | 196171 | 642 | - | Type III effector |
Out of eleven primers, five primer pairs; XCI_1F/R, XCI_2F/R, XCI_3F/R, XCI_5F/R and XCI_6F/R were amplified with expected PCR amplicons of 495 bp, 503 bp, 612 bp, 665 bp and 468 bp, respectively in the
(Fig. 3, Supplementary Table S1). Those amplifications were completely absent in other
We identified the orthologous genes and their genomic neighborhood by EDGAR tool. In addition, we identified only the 5 orthologs genes of
A PCR sensitivity test for
(Fig. 4) and indicated detection limit by PCR.
Comparative genomics is an easy and accessible approach, which provides an opportunity for in-depth analysis of intr-aspecies diversity by using the available genome sequences of different strains in the public data bases. Pangenome of bacterial populations has been used since 10 years ago for comparative genomics; currently, several bioinformatics tools have been developed for comparative genome analysis. However, a fewer online tools is available for comparative genome analysis, especially for bacterial genomes. Among them, EDGAR 3.0 is one of the most popular webserver based tool for the comparative bacterial genome study (Blom
Several molecular methods are used in detection of closely related pathogenic bacterial strains that affecting various crops (Berg
T3E protein, a crucial virulence factors in
Four primer pairs (XCI_1F/R, XCI_3F/R, XCI_5F/R and XCI_6F/R) were able to detect genomic DNA at a low limit of 0.00007 ng/µL (Fig. 4) which an agreement with the dilution test in detecting
Bacterial blight disease is an important disease of many crops or plants which is caused by
The authors have all ethical responsibilities of this manuscript. This research has conducted without any commercial or financial benefits, the authors also declared no conflict of interest and none of the animal and/or human trials were included.
We thank Joana G. Vicente, University of Warwick, UK for providing different strains with known races of
I-SN, J-IP and H-TK conceived and designed the study. DMIJ conducted the
This work was supported by the Center for Horticultural Seed Development (Golden Seed Project No. 213007-05-5-SB510) of the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea.
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