ABSTRACT
Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen is needed for effective disease management. The genome of the begomovirus and betasatellite were amplified in symptomatic plant samples using specific degenerate primers. DNA-A genome of twelve isolates representing different locations in Sri Lanka were cloned, sequenced and deposited in GenBank database (Accession No- KX698087- KX698092 and MH455207- MH455212). Size of the complete nucleotide sequences ranged from 2735 to 2786 bp. The genome organization showed characteristics of begomoviruses. The pairwise sequence identity revealed the association of two different begomovirus species. Five of the isolates showed > 91% of sequences identity with Bhendi yellow vein mosaic virus, and the rest of the seven isolates were around 92% of identity with Okra enation leaf curl virus. This is further supported by phylogenetic analysis where both of these group of isolates were in different cluster. Recombination analysis showed the presence of recombinant fragments in the virus isolates associated with okra yellow vein mosaic disease (OYVMD) in Sri Lanka. Attempts to amplify DNA- B were failed in any of the samples tested. However, both type of the begomovirus species associated with betasatellite species, Bhendi yellow vein mosaic betasatellite. The present study has revealed the association of two distinct monopartite begomovirus species, Bhendi yellow vein mosaic virus or Okra enation leaf curl virus, with OYVMD in Sri Lanka.
Subject(s)
Abelmoschus/virology , Begomovirus/genetics , Plant Diseases/virology , Abelmoschus/genetics , Begomovirus/isolation & purification , Begomovirus/pathogenicity , Cluster Analysis , DNA, Viral/genetics , DNA, Viral/isolation & purification , Genetic Variation/genetics , Genome, Viral/genetics , Phylogeny , Plant Diseases/genetics , Sequence Analysis, DNA , SoftwareABSTRACT
In addition to a unique tumor-inducing (Ti) plasmid, the plant pathogenic bacterium Agrobacterium tumefaciens has an unconventional chromosomal organization. Our previous studies on A. tumefaciens MAFF301001 revealed that it possesses a 2 Mb linear and a 2.8 Mb circular chromosome plus a 206.479 kbp Ti plasmid (pTi-SAKURA). In this study, a linkage map for the left half of its linear chromosome covering a 900 kbp region was constructed and the number of potential genes existing in the region was estimated. The linkage map consists of 31 BAC and 8 lambda phage recombinants without any gaps. It confirmed the size and all the structural landmarks indicated in the corresponding region of our previously constructed physical map for the linear chromosome. Sequencing analysis of the end-regions of each linking clone led to the identification of 6 genes and another 27 potential genes or ORFs, including genes and/or gene clusters responsible for homologous recombination (ruvB), trehalose/maltose sugar transport (thuR, thuG) and alanine catabolism (dadR). Two virulence-related gene homologues (attK and celB), previously reported in the circular chromosome of a different strain of A. tumefaciens were found in this region. These findings will provide a ready-to-use linkage map for further functional analysis of the linear chromosome.
Subject(s)
Agrobacterium tumefaciens/genetics , Chromosome Mapping , Chromosomes, Bacterial , Genome, Bacterial , Agrobacterium tumefaciens/pathogenicity , Alphaproteobacteria/genetics , Bacteriophage lambda , Chromosomes, Artificial, Bacterial , Contig Mapping , Escherichia coli , Genomic Library , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Virulence/geneticsABSTRACT
Genome of A. tumefaciens contains a linear and a circular chromosome. As an initial step of elucidating the structural and functional genomics of this bacterium, linkage map of the left region of its linear chromosome was constructed. Total genomic libraries of A. tumefaciens MAFF301001 were constructed in BAC vectors namely, pFOS1 and pBeloBAC11. Upon construction of sub-libraries, minimum overlapping clones needed to cover the left region was determined. So far, four contigs have been assembled with a total of 19 overlapping clones. Detailed EcoRI physical map of contig III was constructed and it covers a 110 kb region of the Pme5 fragment of the linear chromosome. Seven end regions of the linking clones were partially sequenced but no gene existence was determined due to low homology.
