In planta cloning of geminiviral DNA: the true Sida micrantha mosaic virus.
The circular single-stranded DNAs of geminiviruses are multiplied efficiently and preferentially by rolling circle amplification (RCA), and can be diagnosed readily by restriction fragment length polymorphism (RFLP) and direct sequencing of the RCA product. Two strategies are described for cloning geminiviruses from plants harboring mixed infections by using RCA and RFLP with plant-derived nucleic acids without the need for bacterial amplification. By combining both these approaches, the true Sida micrantha mosaic virus was identified. The advantages of maintaining the quasispecies nature of a virus during in planta cloning is discussed with respect to reliable virus identification and resistance breeding.
Jeske
Holger
H
Biologisches Institut, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany. holger.jeske@bio.uni-stuttgart.de
Gotthardt
Diether
D
Kober
Sigrid
S
D053839:Begomovirus / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular / Q000379:methods*; D016172:DNA Fingerprinting; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D005838:Genotype; D019660:Malvaceae / Q000821:virology*; D010935:Plant Diseases / Q000821:virology*; D012150:Polymorphism, Restriction Fragment Length
D004279:DNA, Viral