Development of Molecular Marker through Genome Realignment for Specific Detection of Xanthomonas campestris pv. campestris Race 5, a Pathogen of Black Rot Disease원문보기
Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned...
Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.
Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.
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문제 정의
Our research group has already developed race-specific molecular markers for Xcc race 1, 3, and 4 [22, 23] and some others are under development. Therefore, the objective of this study was to also develop a marker for rapid detection of Xcc race 5.
oryzae (Xoo) and Xcc [21-23]. Thus, one of our research goals is to identify and determine the predominant Xcc races in Korea. Our research group has already developed race-specific molecular markers for Xcc race 1, 3, and 4 [22, 23] and some others are under development.
제안 방법
Since the genome of Xcc race 5 was not yet sequenced, hence, to develop a race 5-specific molecular marker, we looked for the highly variable genomic regions among the 12 realigned genomes and identified the variable genomic fragment ‘XccR5-89.2’ (Fig. 1).
대상 데이터
The selected positive clones were further cultured in liquid LB media for overnight and plasmid DNA was purified using a commercial kit (GeneAll, Korea). The purified plasmid DNA samples were sequenced by ABI 3730XL DNA Sequencer (Macrogen Inc., Korea) with universal primers. The sequence alignment was performed using Clustal Omega (https://www.
, Korea) with universal primers. The sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).
The whole genome sequences of Xcc races (race 1, 3, 4, and 9), Xcc strains (ICMP 4013, ICMP 21080 and Xcc strain 17) and Xc subspecies (Xci strain ‘CFBP 1606R’, Xcr ‘756C’, Xcv strain ‘85-10’ were downloaded from the NCBI (https://www.ncbi.nlm.nih. gov/genome/microbes/) (Table 2).
성능/효과
S3). Furthermore, the BLASTP was accomplished against the NCBI and found that it has significant homology with the protein encoding an IS4 family transposase of Xanthomonas campestris (WP_011038666.1).
The PCR results showed that the ‘XccR5-89.2’ primers amplified an approximately 2,200-bp fragment specific to Xcc race 5 as well as a 1,515-bp and an over 3,000-bp fragment for race 1 and race 3, respectively (Fig. 2).
The potentiality of race 5-specific primers was determined by bio-PCR with artificially inoculated cabbage leaves with different Xcc races (races 1–8). The results revealed that only cabbage samples inoculated with race 5 were amplified while no visible bands were detected in the remaining 21 samples inoculated with other Xcc races (Fig. 6).
The sequence analysis of the ‘XccR5-89.2’ fragment revealed that it has a 990-bp ORF which showed 100% identity with IS4 family transposase of Xanthomonas campestris.
참고문헌 (40)
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