1a. Objectives (from AD-416):
1. Develop qPCR probes for the different Dekopon sub-isolates and quantify each in single and mixed infections to assess interference in accumulation/replication of each sub-isolate. 2. Examine P20, P23, and CP (suppressors of gene silencing) for each sub-isolate for genetic variation and associate with symptom expression. 3. Characterize siRNA profiles in mixed and single infected plants by high throughput sequencing.
1b. Approach (from AD-416):
1. Aphid transmission 2. Reverse transcription (RT) polymerase chain reaction (PCR) 3. Real time PCR (qPCR) 4. Cloning, sequencing 5. Western blots, northern blots, hybridizations 6. Small RNA deep sequencing 7. Bioinformatics Documents SCA with the University of Iowa.
3. Progress Report:
Results from this study are in support of Objective 3.B of the parent project. During FY12, differential host response and gene expression of symptomatic and asymptomatic citrus plants infected with mixtures of a mild and a severe strain of Citrus tristeza virus (CTV) were examined. Forty families of microRNAs were identified as differentially expressed in symptomatic versus asymptomatic (cross-protected) infected plants. Because previous research on this project had established, genes in the CTV resistance locus (that confers CTV resistance) may play a role in cross-protection, a large sequence database (165 Mb) was produced for sour orange and assembled to generate a draft genome. This genomic information is needed to examine profiles of small viral RNAs in libraries from sour orange plants infected with CTV strain mixtures to identify genetic loci involved in host response to virus infection.