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United States Department of Agriculture

Agricultural Research Service

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Research Project: Further Characterization of Huanglongbing (Hlb) Resistant Clones of Selected Varieties

Location: Subtropical Insects and Horticulture Research

Project Number: 6618-21000-014-21
Project Type: Reimbursable

Start Date: Nov 27, 2013
End Date: Apr 30, 2015

Objective:
The objective of this project is to verify resistance to huanglongbing in three genera of citrus relatives that are cross-compatible with cultivated citrus and identify genes associated with this resistance in the original species and their hybrids.

Approach:
Species of Poncirus, Microcitrus and Eremocitrus and some of their hybrids have shown promising tolerance/resistance to Huanglongbing (HLB) in a recently concluded field trial. Replicated seedlings of species from these three genera, their hybrids with cultivated citrus, and susceptible cultivated citrus parents will be grown in the greenhouse. At six months plants will be inoculated with the HLB pathogen Liberibacter Asiaticus (LAS) using infected psyllid vectors. At 1, 3, 6 and 12 month intervals, sampling will be done as follows. Two control plants and six inoculated plants will be uprooted, washed, blotted and air-dried. Leaf midrib samples from upper, middle and lower parts of the plant will be collected separately from each plant. Two samples will be collected from roots. A total of 5 samples per plant, and a total of 440 samples will be collected at each time point. Each sample will be collected in 2 ml screwcap tubes containing RNAlater solution and stored frozen for DNA and RNA extractions. DNA extractions will be analyzed for the presence of LAS. DNA extractions of plants showing differential response to HLB will be analyzed for host genome diversity by simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) analyses to verify genotypes. RNAseq will be conducted on Microcitrus and Eremocitrus with and without LAS infection at one month after inoculation. Gene expression will be analyzed for resistance candidate genes from RNAseq of these genera and for genes identified by other researchers as associated with HLB-resistance (including Poncirus hybrids). Specifically, we will first target genes reported as having a role in resistance: genes associated with cell organization and biogenesis (expansin family and endo- 1,4-ß-glucanase), genes involved in cell wall degradation, protein metabolism (leucin-rich repeat protein kinases), protein degradation, disease resistance genes, gene known to be expressed during abiotic or biotic stress, genes coding for transcription factors, pathogenesis related proteins, etc. (Fan et al., 2012; Albrecht and Bowman, 2012). We will study gene expression using real-time quantitative polymerase chain reaction (qPCR) technique, and use conventional PCR and sequencing where necessary. We will also plant 10 seedlings of each accession used for the greenhouse study in field to compare resistance in these genotypes under conditions employed in the initial field experiment. This will also provide additional infected materials for analysis.

Last Modified: 10/1/2014
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