PCR and RFLP analyses based on the ribosomal protein operon

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene. RFLP analysis of 16S rRNA gene sequences has identified 31 16S rRNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can, however, become more refined and specific if moderately conserved genes, such as the ribosomal protein (rp) genes, are used as genetic markers. The use of additional genetic markers enhances the resolving power of phytoplasma classification. This chapter describes the methodology of detection, differentiation, and classification of phytoplasma strains based on rp gene sequences. RFLP analysis of amplicons obtained by group- or subgroup-specific rp gene-based primers is used for finer differentiation of phytoplasma strains within a given group or subgroup. The rp gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains. Many individual 16Sr subgroups can be further differentiated into two or more distinct rp subgroups.