Multilocus Sequence Analysis for Revealing Finer Genetic Variation and Phylogenetic Interrelatedness of Phytoplasma Strains in 16SrI Group in China

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Objective The phytoplasmas in 16SrI group cause severe diseases of many crops and ecological plants in China. At present, the genetic variation and population structure of the yet-uncultured and very closely related phytoplasma strains in China are still not fully understood. A multilocus sequence analysis (MLSA) scheme was used to elucidate genetic diversity of phytoplasma strains in 16SrI group from different regions in China and their relationships with geographical distribution, and to compare the levels of variation in different housekeeping gene of different phytoplasma strains. The study aimed to provide some reference approach and evidence for finer detection, identification, classification and phylogenetic analysis of different phytoplasma strains in China. Method Ten housekeeping gene (rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB) fragments combined with 16S rDNA sequences were employed to analyze the genetic variation and phylogenetic relationships of 18 phytoplasma strains infecting chinaberry, lettuce, mulberry, paulownia and periwinkle from ten provinces in China, using five phytoplasma strains, onion yellows phytoplasma (OY-M), aster yellows witches'-broom phytoplasma (AYWB), Candidatus Phytoplasma australiense (CPA), strawberry lethal yellows phytoplasma (SLY) and Candidatus Phytoplasma mali (CPM), of which whole genomes have been sequenced, as references. Sequence polymorphism and variation levels of different gene fragments were analyzed by multiple sequence alignment. Result The nucleotide site polymorphisms of rp, tuf, secA, secY, ipt, dnaK, fusA, gyrB, pyrG and rpoB gene fragments were used to resolve all strains into 15 sequence types (STs), demonstrating extensive genetic diversity among the 16SrI group strain population. All the strains, classified into 16SrI-B and-D subgroup by 16S rDNA analysis, were clustered into one clade and clearly differentiated into discrete subclades by phylogenetic analysis of the concatenated gene sequences. Ten chinaberry witches'-broom strains, which were most closely related to two mulberry dwarf strains and hardly distinguished with 16S rDNA, were definitely split into four distinct clusters and 8 STs apparently relative to their geographical origins. Two lettuce yellows strains in Sanming, Fujian province, China, were more closely related to the onion yellows OY-M strain in Japan than the periwinkle virescence and paulownia witches'-broom strains in China. The levels of variation in dnaK locus were higher than those in 16S rDNA and other genes tested. Conclusion It is suggested that the MLSA should potentially be a useful and reliable approach for phytoplasma identification and differentiation as well as for depth examination of strain diversity and this method can be wildly used to study the genetic variation and evolutionary relationships of phytoplasma strains among various groups or subgroups in the future.