Multigene phylogeny of an indian isolate of Radopholus similis infecting black pepper (Piper nigrum L.)

The accurate biological identification and characterization of nematodes infecting a crop is a pre-requisite for designing effective control strategies. Quantifying genetic diversity and species discrimination of Indian population of burrowing nematode, Radopholus similis, was achieved by sequencing a series of marker genes and their phylogenetic analysis. The mitochondrial cytochrome c oxidase gene (coxl), NADH dehydrogenase gene (nd1), internal transcribed spacer region (ITS1–5.8S-ITS2), D3 expansion domain segments, 18S ribosomal RNA gene and 28S ribosomal RNA gene were used for the study. Primers were designed from already deposited sequences from NCBI to amplify, clone and sequence the genes for characterizing R. similis R01 isolate infesting black pepper in India. The generated sequences were deposited in NCBI and their in silico characterization revealed that the sequences were highly similar to their counterparts from other countries. A comparative inter-specific analysis of each marker genes along with the genes from GenBank revealed that polymorphism, divergence and conservation are more expressed in mitochondrial segment for R. similis. Phylogenetic relationship was inferred using various methods like maximum parsimony, maximum likelihood, Bayesian algorithm and neighbour-joining. The haplotype network analysis of the global burrowing nematode populations using the ITS sequences clearly grouped them into three clusters - the Asian and Australian R. similis populations forming a single cluster, the African R. similis populations another cluster and the R. duriophilus and R. arabocoffeae populations along with the Indian R. similis population R01 forming a distinct third cluster. This study is the first report of using MLST approach by means of concatenated genes for the phylogenetic characterization of R. similis.