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United States Department of Agriculture

Agricultural Research Service

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Research Project: Screening and Cloning of Resistance Relates Genes by Rna-Seq in Huanglongbing (Hlb) Resistant and Susceptible Citrus Breeding Lines

Location: Subtropical Plant Pathology Research

Project Number: 6034-22000-039-27
Project Type: General Cooperative Agreement

Start Date: May 01, 2012
End Date: Mar 31, 2015

Objective:
1. To generate transcriptome profiles of both susceptible and resistant citrus responding to huanglongbing (HLB) infection using RNA-Seq technology. 2. To identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as real-time polymerase chain reaction (RT-PCR). 3. To create transgenic citrus cultivars with new constructs containing the resistant gene(s).

Approach:
1. Extract mRNA from four susceptible and four resistant plants from each of three progenies (24 genotypes) and use next generation sequencing technology to sequence all mRNA. 2. Clean the reads from RNA-Seq and assemble the RNA-Seq reads to generate transcriptome. 3. Identify differentially expressed genes using statistical tests. 4. Annotate differentially expressed genes. 5. Use random matrix theory-based method to identify differentially expressed gene networks. 6. Use real time polymerase chain reaction (PCR) to verify the differentially expressed genes in original 24 genotypes. Consistency of these resistance/susceptibility genes will be further verified through evaluation of 24 additional highly resistant and susceptible genotypes from more diverse genetic backgrounds. 7. Most promising genes will be used in citrus-only constructs and used to create transgenic citrus.

Last Modified: 12/29/2014
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