ARS | Publication request: Novel Molecular Technique for Rapid Cloning of Unknown Sequences From Unculturable Huanglongbing (HLB) Associated Bacterium, Candidatus Liberibacter

Submitted to: American Phytopathology Society
Publication Type: Abstract
Publication Acceptance Date: March 15, 2007
Publication Date: July 28, 2007
Citation: Lin, H., Doddapaneni, H., Liao, H., Bai, X., Zhao, X., Wang, J., Civerolo, E.L. 2007. Novel Molecular Technique for Rapid Cloning of Unknown Sequences From Unculturable Huanglongbing (HLB) Associated Bacterium, Candidatus Liberibacter. American Phytopathology Society. 97(7):64

Technical Abstract: Huanglongbing (HLB), previously known as citrus greening disease, is one of the most destructive citrus diseases worldwide. Recent occurrence of citrus Huanglongbing(HLB)in Florida has dramatically increased the threat to the entire US citrus industry. The disease is associated with Candidatus Liberibacter (Ca. L.) spp., an unculturable, phloem-limited bacterium for which limit genomic information is available. We have developed an effective PCR-based DNA walking approach using a shorter strand adaptor modified with a blocking group at the 3¿ and suppression PCR technique. This approach has resulted in 100 percent efficiency for obtaining Liberibacter target DNA sequences from mixed DNA samples. A total of 8,813 bp of new DNA sequences were obtained from three genomic loci; rpoBC gene cluster, omp gene cluster and 16/23S rRNA gene region in Ca. L. asiaticus. These, together with other publicly available sequences, make up to 24.4 Kb of genomic sequence. BLAST annotation predicts 12 full length genes, 2 partial genes and one pseudogene among these sequences. Comparative genomics and phylogenetic analyses of the newly cloned genomic regions were conducted for better understanding the genome characteristics and taxonomic position of Ca. L. The sequences obtained in this study will facilitate development of new genome-based detection tools. The technique described here can also be employed to acquire new genomic information for other unculturable or fastidious organisms with limited sequence information or for filling gaps between known flanking genomic DNA sequences.