The Spatial And Temporal Distribution Of Apple Scar Skin Viroid And Its Effects On Anthocyanin Synthesis And Related Genes

Apple Scar Skin viroid is a great harm to fruit trees. Recently, Apple Scar Skin viroid has a tendency to spread in our major apple producing regions, which restricts the healthy development of Chinese apple industry seriously. Currently, there is little knowledge about the occurrence of ASSVd, and there is no effective prevention and control measures. In this study, the detection system of ASSVd was established and optimized by using real-time PCR techniques. And in order to provide a basis for the detection and prevention of ASSVd, we studied the spatio-temporal distribution of ASSVd in apple trees with this detection system. Meanwhile, the effect of ASSVd on the anthocyanin synthesis and the transcribed levels of related genes was researched in order to acquaint the pathogenesis of ASSVd. The main results were as follows:1.The establishment and optimization of real-time PCR method to ASSVdThis research established ASSVd real-time PCR detection method and validated the specificity, sensitivity, applicability and reproducibility of this method. The study demonstrated that:(1)The sensitivity was high. The real-time PCR sensitivity was 100 times higher than conventional RT-PCR.(2)The specificity of primers was strong. It could detect ASSVd, while it had no use on ACLSV, ASGV and ASPV.(3)The applicability was wide. It can detect the ASSVd in flower, leaf, bark and root or other different tissues(4)The reproducible was good. Measuring 3 concentrations of c DNA templates, the high, medium, and low concentrations were 100、10-1、10-2 respectively. The variation coefficients of Ct values were within the error range after repeating 3times. And this study optimized the method by designing different primer concentration and annealing temperature, eventually, it proved that the 10μmol/L of primer concentrations and annealing temperature 60℃ were the best.2.The clone and sequence analysis of ASSVd in HebeiUsing five apple varieties(Dounan, Gala, Huashuo, Zhongqiuwang and Fuji) whichappeared the ASSVd symptoms as materials. In this research, we ensured them carrying the ASSVd by the RT-PCR techniques, then we received 14 different sequences after cloning and sequencing. The homology of the 14 different sequences which obtained from the analysis by DNAMAN 6.0 is 98.49%. The results were highly homologous with the ASSVd sequences from Xinjiang, Beijing and Korea which reported in NCBI, the homology is 95.35%. The results showed that the variation of ASSVd sequence had no significant correlation with varieties.3. The temporal and spatial distribution of ASSVdTaking the leaf, bark, root and fruit of Fuji apples carrying ASSVd as materials and using the established ASSVd real-time PCR method to detect. The results showed the differences of ASSVd distribution in the temporal and spatial areas:(1) The relative content of viruses in underlying parts was more than the upper parts;(2)The relative content of virus in young organizations was less than in mature tissues;(3)The relative content of virus in hot season was lower than in cold season. From the overall results, we obtained the result that the relative expression of ASSVd was the highest at roots in December. Therefore, it is credible that collecting the root bark tissue samples in December for detecting.4. The effects of ASSVd on the anthocyanin synthesis and related genes(1) The change of anthocyanin contents showed an upward trend in the healthy apple peels and the peels carrying ASSVd after removing the bags from 0 day to the 14 th day, with the 6 acquisition periods,but the anthocyanin content in healthy peels was higher than diseased peels significantly.(2) In the study on the structure genes of anthocyanin synthesis, we found that the relative expression of Md CHS, Md F3 H, Md DFR, Md ANS, Md UFGT in healthy peels were higher than the diseased peels in the first day after removing the bags. Except Md F3 H, the other four genes of healthy peels were higher than diseased trees in the 2d-14 d after removing the bags. The study on related regulatory genes for anthocyanin synthesis showed that: The relative expression contents of Md BHLH33 and Md MYB11 in healthy apple peels were higher than those diseased peels after removing the bag 0 day significantly, then the expression levels of Md BHLH33, Md TTG1 and Md MYB11 had been lower than in the diseased peels. It suggested that the ASSVd affected the gene transcription of anthocyanin synthesis in apples.(3) The relevance of anthocyanin contents and related genes’ relative expression were different between the healthy apple peels and the peels carrying ASSVd. The genes relative expression content of the Md UFGT and Md DFR in healthy peels was positively correlated with the anthocyanin content, and the relative expression content of Md DFR was positively correlated with the anthocyanin content, remarkablely,(correlation coefficient is 0.923). While, the other structural genes’ relative expression were all positively correlated with the anthocyanin content and the high correlation coefficient was high. While, only Md ANS structures genes’ relative expression content of diseased peels was positively correlated with anthocyanin content significantly, the others had no significant correlation.