| Citrus tristeza caused by Citrus tristeza virus(CTV)is a disease that seriously threatens the green development of citrus.The disease has killed hundreds of millions of citrus trees worldwide.To clarify the relationship between etiology and disease and effectively prevent this disease,it is necessary to have a deep understanding of the population composition of CTV pathogens in citrus decline disease,and to use efficient detection and identification of CTV genotypes.This is the basis for achieving disease control and green citrus development.The existence of new isolates and their molecular and biological characteristics in CTV populations was clarified,which provided a theoretical basis for the prevention and control of CTV pathogens.Efficient and accurate identification of CTV genotype composition,providing technical support for better prevention and control of CTV.In this study,a novel CTV isolate was identified on Wild Orange in Daoxian and the full sequence of the isolate was obtained.At the same time,a highly efficient genotype multiplex RT-PCR identification method was established,and the results are as follows1.Identification of new CTV genotypes: Metrovirus sequencing with RT-PCR and RACE-PCR validation obtained the whole genome sequence of one Daoxian wild orange CTV strain(JY-2),with a total genome length of 19112 nt,registration number ON094625.Homologous sequence and phylogenetic analysis of reported CTV genotype isolates and JY-2 showed that JY-2 was clearly different from other known CTV genotypes and existed independently.The genome-wide similarity was less than 92.5 %,while the ORF1 a nucleotide was less than 92 % similar to the amino acid sequence.It is speculated that JY-2 may be a new CTV genotype,named JY genotype.2.The aim was to identify the genotype of JY and to establish a new genotype detection system applicable to CTV.For this purpose,we designed a set of primers whose design was based on the nucleotide variability of the p6 region.Subsequently,we performed RT-PCR verification.Eventually,this new genotype detection system was established.The JY genotype detection system was used to test citrus samples from 9 main cultivation areas and 4 wild citrus areas,and only 12 positive samples were detected in Citrus daoxianensis.12 JY isolates were analyzed by molecular cloning of their isolate’s ORF1 b,p33,p25 and p23 gene sequences,and sequence homology analysis with the corresponding gene sequences of reported CTV genotype isolates,genetic.The sequences were analyzed for sequence homology,genetic differentiation and population dynamics,molecular variation,primary genetic analysis and phylogenetic analysis.The results showed that the sequence similarity among the four genes of JY isolates was high,indicating that the genetic variation within the JY genotype population was low and the population was stable.there was obvious genetic differentiation between the JY genotype and other CTV genotypes,and the differences between the CTV wild citrus population and the cultivated citrus population were very large and the gene exchange was small;the JY isolates clustered in the same cluster and were independent of the known CTV genotypes.3.Construction of CTV genotype multiplex RT-PCR identification system: The reference sequences of CTV genotypes were downloaded from NCBI,and primers were designed at positions with obvious differences in nucleotide sequences,and the ideal primers for each genotype identification were screened by specificity,generality and sequencing verification.Two sets of multiplexed genotype identification systems were designed by combining the selected primers according to the amplification fragment size and the non-interference between the primers.By adjusting and optimizing the system parameters,two stable multiplex identification systems were finally obtained.The sensitivity of the multiplex systems was tested with 10-fold gradient dilutions of the plasmids.The sensitivity of the two systems was found to be 1.65 μg / μL,and the results of the single genotype identification system were compared with those of the multiplex system,and the genotype identification results were consistent. |
