Detection of apple latent spherical virus in seeds and seedlings from infected apple trees by reverse transcription quantitative PCR and deep sequencing: evidence for lack of transmission of the virus to most progeny seedlings

作者：Kishigami Ryusuke; Yamagishi Noriko; Ito Tsutae; Yoshikawa Nobuyuki^*

来源：Journal of General Plant Pathology, 2014, 80(6): 490-498.

DOI：10.1007/s10327-014-0541-3

• Apple latent spherical virus
• Seed transmission
• RT-qPCR
• Deep sequence analysis

We used reverse transcription quantitative PCR (RT-qPCR) to test for apple latent spherical virus (ALSV) in cotyledons of seeds and true leaves of seedlings from apple trees infected with ALSV to accurately determine the status of infection in seedlings from ALSV-infected trees. The amplification curves of ALSV-RNAs from cotyledons were classified into three patterns: fast (F), slow (S), and no amplification (N/A). Viral RNA was detected in high concentrations (F pattern) in cotyledons from approximately 1 % of the seeds, a proportion broadly consistent with previously reported ALSV seed transmission rates (from zero to a few percent). In the majority of seeds (94.4 %); however, minute amounts of RNA related to the virus were detected (S pattern). Minute amounts of viral-related RNA were also detected by siRNA analysis using a next-generation sequencer. After the seeds germinated, however, no viral RNA was detected in the true leaves, so we concluded that the viral RNA in the seeds lacked the capacity to replicate. No ALSV-RNA at all was detected (N/A pattern) in the remaining 4.6 % of seeds. We concluded that almost all apple seedlings from ALSV-infected trees (approx. 99 %) can be considered as virus-free. Therefore, we think that apple seedlings from the generation obtained after the use of ALSV vector technology can be exempted from restrictions on genetically modified plants after they have been confirmed to be virus-free by virus testing.

• 出版日期2014-11