Location: Subtropical Plant Pathology Research2013 Annual Report
1a. Objectives (from AD-416):
1. Generate transcriptome profiles of both susceptible and resistant citrus responding to huanglongbing (HLB) infection. 2. Identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants. 3. Create transgenic citrus cultivars with new constructs containing the resistant gene(s).
1b. Approach (from AD-416):
Using RNA-Seq with next generation of sequencing technology, and intensive bioinformatics to identify key resistant genes from susceptible and resistant citrus plants in response to Candidatus Liberibacter asiaticus infections, and using routine transformation techniques to create transgenic citrus plant with the select resistant gene.
3. Progress Report:
This project is related to in-house project objective: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens. First group of 5 samples for RNA-Seq (also called "Whole Transcriptome Shotgun Sequencing"), including resistant/tolerant vs. susceptible plants were nearly completed. Second group of 10 samples is in the sequencing process. We mapped the RNA-Seq data to reference genome, Citrus clementine using the computer program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24275 genes of original predicted genes had been found with expressions. And a total of 10539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the Nucleotide Binding Site (NBS) genes were found to have an expression. For Citrus clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or Expressed Sequence Tags (EST) s deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of EST varied from 1 to 25. We identified a few Leucine Rich Repeat Protein-Kinase (LRR-PK) genes for further comparative study based on the RNA-Seq data. The results indicated sequence variations of these genes in different varieties are indeed due to Single-Nucleotide Polymorphisms (SNP)/indels, and some of them were annotated as putative pseudogene because of a truncation. Further verifications is underway.