Small RNA sequencing of Scutellaria baicalensis reveals brassica yellows virus infection and the complementary development of a sensitive and reliable RT-qPCR assay. (April 2023) - Jin, Weijie
Abstract: To identify the potential causal agent that has caused Scutellaria baicalensis to exhibit yellowing and other morbid symptoms, we sampled from this medicinal plant in Gansu Province, Northwest China, namely, that plant's main growing area. All 18 contings obtained from small RNA sequencing (sRNA-Seq) were homologous to brassica yellows virus (BrYV), with 93%–100% nucleotide similarity. A full-length genome (5693 bp) was obtained using segmentation amplification and Rapid Amplification of cDNA Ends (RACE), which was subsequently identified as BrYV-HQ. Genomic similarity between BrYV-HQ and other isolates ranged from 80.64% to 90.58%. Observed variation was mainly in the coding region of the ORF 3–5 fusion proteins. Evolutionary tree analysis indicated that the kinship of BrYV-HQ may be linked to BrYV-NtabQJ (MK057527.1), known to infect tobacco plants produced in China. Recombination analysis indicated that BrYV-HQ may have derived through recombination between BrYV-NtabQJ and a BrYV isolate (KY310572.1) that infects Brassica napus in China. Based on the coat protein (CP) gene design of the RT-qPCR primer set obtained, the linearity of the standard curve equation y = −2.987*Log(x) + 41.75, which we established for quantitative detection, was good (ranging from 4.43 × 10 10 copies/μl∼4.43 × 10 3 copies/μl) for the quantitative detection of BrYV-HQ. This study marks the first time that BrYV has been confirmed to infect S. baicalensis and upon which a quantitative detection method was established, providing a molecular basis for the research and management of S. baicalensis viral disease in China. Highlights: First report of brassica yellows virus infecting Scutellaria baicalensis in China. Complete genome sequence analysis identifies a new recombinant brassica yellows virus. Established a rapid detection system using RT-qPCR to detect brassica yellows virus.