Effect of in vitro sub-culture of sandy pear on variant structure of apple stem grooving virus

Abstract

In vitro culture of sand pear (Pyrus pyrifolia) cv. Huanghua (HH) infected with apple stem grooving virus (ASGV) was conducted and the titer and variant mutations of the virus in sub-culture were analyzed. The results showed that ASGV could replicate stably in 20 successive sub-cultures. The 500 bp PCR products of ASGV in each sub-culture were cloned and used for single-strand conformation polymorphism analysis. The results demonstrated that there was a dominant molecular variant of ASGV in vitro and that the variant structure of ASGV is stable in sub-culturing. The rate of the main haplotype in the 20 passages was from 78.0% to 95.7%. To clarify the origin of subordinate haplotypes, the error rate of our RT-PCR assay (5.4× 10−4/nt) was computed and the results indicated that the mutations which appeared in sub-passages were acquired from artificial operation and not produced by viral replication.