High-throughput Sequencing (HTS)-Based Diagnosis of Geminiviruses.

The development of high-throughput sequencing (HTS) technologies has revolutionized plant virology. One of the many application of HTS is the detection and diagnostics of plant viruses. The main advantage of HTS compared to other diagnostics methods is its unbiased nature, meaning that it is capable of detecting any virus present in the sample.

Cloning of Complete Geminivirus Genomes by Rolling Circle Amplification (RCA).

Rolling circle amplification (RCA) revolutionized geminivirology before high-throughput sequencing. Using the DNA polymerase of phage phi29 and random primers, it is a simple isothermal reaction which will amplify circular DNA without any prior knowledge of its sequence. It is thus ideal to clone complete geminivirus genomes.

Mechanical Sap Transmission of Geminiviruses.

Mechanical sap transmission is the simplest, quickest, and most cost-effective method to transmit geminiviruses (and indeed any plant virus) from one plant to another. However, a significant number of geminiviruses, which are phloem-restricted in their hosts, are not transmitted in this form. For those geminiviruses which do invade the mesophyll, mechanical transmission is possible.

Extraction of Geminivirus and Associated Satellite DNA from Leaf Samples.

The isolation of DNA from geminiviruses and their associated DNA satellites in plant leaf samples is a key step, as high-quality DNA preparations are essential for studies related to the characterization of geminivirus genomes and their interactions with host plants. The protocol outlined in this chapter describes a methodology based on the use of cetyltrimethylammonium bromide (CTAB) that facilitates the efficient extraction of total nucleic acids from plant tissues. The DNA obtained through this protocol finds direct application in various molecular techniques such as polymerase chain reaction, Southern blot, dot blot, and rolling-circle amplification.