Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

Abstract

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

Keywords

• strawberry virus
• brassica yellows virus
• complete genome sequence
• infectious clone
• TaqMan RT-qPCR