ARS | Publication request: Development of SSR Markers for Detection, Genotyping, and Genetic Diversity Assessment of “Candidatus Liberibacter asiaticus”

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 30, 2009
Publication Date: August 1, 2009
Citation: Lin, H., Doddapaneni, H., Chen, C., Duan, Y., Zhou, L. 2009. Development of SSR Markers for Detection, Genotyping, and Genetic Diversity Assessment of “Candidatus Liberibacter asiaticus”. Phytopathology(99):S74.

Technical Abstract: Huanglongbing, previously known as citrus greening, is a destructive disease of citrus. The causative pathogen is believed to be a phloem-restricted bacteria, “Candidatus Liberibacter” which is naturally transmitted by the citrus psyllid, Diaphorina citri in Asia and America, and Trioza erytreae in Africa. Taxonomically, there are three Liberibacter species associated with citrus Huanglongbing; “Ca. Liberibacter asiaticus” (Las), “Ca. Liberibacter africanus” (Laf) and “Ca. Liberibacter americanus” (Lam). Among them, Las is the most widely spread in the citrus growing regions of Asia and America. Due to the as yet un-culturable nature of the agent, information regarding the pathogen’s genetic variation, population structure, epidemiological relationships related to its evolutionary adaptation, and host selection is very limited. Using the recently sequenced whole genome of Las, we conducted a genome-wide search to identify simple sequence repeat (SSR) sequencing loci and developed a multi-locus SSR marker system for Las genotyping and genetic analyses. Thirty five loci were identified which contain various types of repeat motifs suitable for SSR primer design. Among them, 6 SSR primers showed polymorphisms after validation against multiple Las strains from China, India, Brazil and Florida. Computational algorithms were conducted to carefully design multiplex primer sets that allowed combining 2, 3 or up to 4 sets of primers each labeled with a different fluorescent dye (FAM, NED, PET and VIC) for simultaneous amplification in a single PCR reaction. This multiplex detection platform will increase through-put of sample analysis and is suitable for a large scale genotyping, genetic diversity and molecular epidemiological studies of HLB.