ARS | Publication request: Metagenomic analysis of Candidatus Liberibacter asiaticus in naturally populated psyllids (Diaphorina citri) using BAC libraries

Submitted to: American Phytopathological Society
Publication Type: Abstract Only
Publication Acceptance Date: March 16, 2011
Publication Date: N/A

Technical Abstract: Candidatus Liberibacter asiaticus (Las) is the most prevalent species of three species of Ca. Liberibacter causing citrus huanglongbing (HLB) in the world. The Las genome sequence published in 2009 was obtained from a single Las-infected psyllid using metagenomic approach. Studies of genetic diversity suggest a strong potential of various populations of Las bacteria exiting in different hosts. In this study, three BAC libraries were constructed using whole genomic DNA from thousands of Diaphorina citri collected from the HLB-affected citrus plants in the fields. A total of 61,440 clones were obtained from three libraries constructed by partial digestions of BamHI and HindIII, respectively, or random shear. Superpools and pools of DNA from the BamHI BAC library were screened by conventional PCR using Las-specific primers. Thirty sets of the Las-specific primers were designed with a distance from 30-50kb based on the Las-psy62 genome. Positive BAC clones were identified and subjected to end sequencing. The results indicated 54 overlapping clones that matched the Las genome sequence with a size range from 20kb to 140kb. PCR confirmation of the end sequence using infected vs. non-infected plants or insects with the primer sets designed from the end sequences revealed 7 overlapping clones contained new sequences that were missed in the psy62 genome, while 15 clones did not match the Las sequence, suggesting a potential chimera in these BAC clones.