Barcoding of Plant Viruses with Circular Single-Stranded DNA Based on Rolling Circle Amplification

Abstract

The experience with a diagnostic technology based on rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analyses, and direct or deep sequencing (Circomics) over the past 15 years is surveyed for the plant infecting geminiviruses, nanoviruses and associated satellite DNAs, which have had increasing impact on agricultural and horticultural losses due to global transportation and recombination-aided diversification. Current state methods for quarantine measures are described to identify individual DNA components with great accuracy and to recognize the crucial role of the molecular viral population structure as an important factor for sustainable plant protection.

Keywords: Circomics; RCA; RFLP; geminivirus; nanovirus; satellite.

Figure 1

African Cassava Mosaic Virus TempliPhi rolling circle amplification (RCA) product spread on a cetyltrimethylammonium bromide (CTAB)-activated carbon film, contrast is obtained by Pt evaporation, shown at two magnifications (bar = 1 µm). Note the pinwheel structures, indicative of multiple-primed circular DNA (asterisk).

Figure 2

Steps of RCA on geminiviral single-stranded (ssDNA). Primers may be RNA or DNA of plant origin, or random hexamer phosphorothioate-protected primers added to the reaction mixture. Colours: red viral strand, blue complementary strand. Similar intermediates are generated in vivo during geminiviral replication modes, in CSR, RCR, or RDR.

Figure 3

Statistics of the restriction enzyme recognition sequence AATT (for MluI) for the indicated genera and all others (rest). Left column for fragment size, right column for distribution by number of fragments for the indicated genera based on the data as shown in Table 1. Stippled lines indicate the expected value, calculated on the basis of the nucleotide frequencies. y axis: relative frequency; size in bp. The whole data set for all four nucleotides recognition sites is given in Figure S1.

Figure 4

Statistics of the restriction enzyme recognition sequence AATT (for MluI) as in Figure 3, but relating fragment sizes to genome position. Dot intensity indicates frequency. The whole data set for all four nucleotides recognition site is given in Figure S2.

Figure 5

Accuracy of the fragment size determination. RCA/RFLP (HpaII) of various geminivirus samples (Abutilon mosaic virus, AbMV; tomato golden mosaic virus-common strain, TGMV-cs; beet curly top virus, BCTV; Sida golden mosaic Costa Rica virus, SiGMVCRV; African cassava mosaic virus, ACMV; tomato golden mosaic virus-yellow vein, TGMV-yv) are compared with an AbMV standard (mixture of PstI- and HpaII-cut RCA products), mock-inoculated (m), and undigested (ud) of AbMV RCA product. Separation was performed in a 2% agarose gel and staining with ethidium bromide (inverse image presentation). The positions of genomic fragments (circles) were determined using Fiji (Image J), the corresponding migration distances in numbers of Pixel recalculated in Excel (lower graph) to relate the fragment sizes (in bp) in logarithmic scale to probit values. All data fit to a line with Pearson’s correlation coefficient of R² = 0.991 corresponding to a mean error of 1.6±1.3. Asterisks indicate fragments from defective DNAs of BCTV. Molecular weights (nt) for the standards are indicated on the right, yellow genomic size, red DNA A, green DNA B. Relative migration distance (Rf) values were determined in relation to the start and front positions.