Molecular Biological Detection And Genetic Analysis Of Apple Scar Skin Viroid From Different Apple Varieties

Apple is one of the most widely planted and produced fruits in the world.China’s apple cultivation area and yield rank first in the world.Apple Scar skin virus(ASSVd)is a major viral disease that affects apple growth.It mainly harms apple fruits,causing irreversible changes in fruit phenotype,seriously affecting fruit quality,reducing commodity value,and having a significant impact on the industry.Due to factors such as high heterozygosity and long childhood in the apple genome,as a perennial woody plant,ASSVd adapts to complex host environments through continuous variation.To clarify the molecular variation and phylogenetic patterns of apple rust virus on different apple varieties,this study conducted PCR detection of ASSVd on different apple varieties,and gene cloning was performed on positive samples to explore the population and variant types of ASSVd in different varieties.The aim is to provide theoretical support for the prevention and control of ASSVd and the study of virus host interaction.The main research findings are as follows:1.143 apple varieties were tested for ASSVd by RT-PCR,41 positive varieties were detected,with a incidence rate of 28.7%.2.ASSVd cloning was performed on 41 positive varieties,and a total of 71 effective sequences with 328～333 bp were obtained.Through multiple sequence alignment,17 328～333 bp variant sequences were obtained;The multiple sequence alignment of 17 variant sequences showed that the sequence similarity between different clones obtained from the same sample was 97.28%～99.4%,the similarity of all variant sequences was 84.98%～99.70%,and the similarity with 7 ASSVd nucleotide sequences from different countries and regions registered in NCBI was 98.48%～99.70%;We analyzed the reasons for the formation of 17 variant sequences,mainly including base substitution,base deletion,and base insertion.Among them,base substitution occurs more frequently than base deletion,and base deletion occurs more frequently than base insertion;From the location of base sequence changes,it can be seen that base changes mostly occur in the V and C regions,while other regions such as Tr、TLand P also have relatively few occurrences.3.The infection of different parts of the Venus Golden Apple variety ASSVd was studied,and the results showed that ASSVd infection could be detected on the leaves,branches,bark,roots,and fruits of the same tree.A total of 3 variants were obtained from Venus Gold,with lengths ranging from 328 to 332 bp.The similarity between the different variants was 98.49%to 99.10%.The mutation sequences were mainly generated at sites 148(T→C),94(T→C),202(C→T),219(A→G),220(T→G),and 327～330(missing bases).4.Predict the secondary structure of 17 variants,all of which are rod-shaped structures;The FY-2 and HS variants,which are closely related to MN598207.1,are clustered into Class 1,while the other 15 variants are clustered into another branch.