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United States Department of Agriculture

Agricultural Research Service

Related Topics

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

2013 Annual Report

1a.Objectives (from AD-416):
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).

1b.Approach (from AD-416):
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.

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.

We performed the principal component analysis (PCA) on the expressions of five samples. The plot of first PC against second PC showed that R2017 and R20T18, were grouped to the one group (resistance group) and R19T23, R19T24 and R20T24 were grouped to another group (susceptible group). This result showed that the gene expressions were significantly different in resistant and susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and FDR threshold of 0.01. Among them, 306 genes are up-regulated expression genes and 515 are down-regulated in resistant citrus. Using program iAssembler, a total of 53981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, comparing with the citrus reference genome.

To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612618 SNP/INDELs were identified using mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type mutation is the mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is the mutations in resistant citrus genes that may gain a new function (Type2). Type1 mutation should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which had high impact variations, such as frame shift, splice site acceptor, splice site donor, start lost, stop gain or stop lost and 3578 genes with Type2 mutations. We expect that with more libraries being sequencing, the candidates should be reduced to a reasonable number for further validation.

Last Modified: 4/16/2014
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