Location: Plant, Soil and Nutrition Research
Project Number: 8062-21000-037-05
Start Date: Oct 01, 2010
End Date: Apr 30, 2014
The general approach of this project includes extraction of apple fruit RNA, reverse transcription to cDNA, addition of sample-specific DNA “barcode” sequences, primer adapter ligation to facilitate DNA sequencing and Illumina-based pyro-sequencing of multiplexed samples to generate large numbers of transcript reads from each tissue. Resulting transcript-related cDNA sequence data (approximately 2-4 million reads/sample) will be sorted bioinformatically by barcode sequences to allow assignment to originating tissue samples, assessed for quality and normalized against all other samples to facilitate gene expression assessment based on relative numbers of transcripts per gene. Gene identify will then be assigned based on mapping of all sequence reads to the publically available apple genome sequence. The collaborator’s group is expert in bioinformatic analysis of transcriptome data and in the creation of gene expression and other bioinformatics tools. The ARS lab does not have strong competence in these areas. Genes associated with certain fruit quality traits will be subject to gene-specific primer design for quantitative RT-PCR gene expression analysis to confirm sequence-based expression data. Genes highly correlated with apple fruit disorders will be forwarded to the lab of our ARS collaborators in Washington State for development of marker systems diagnostic for predicting fruit disorders during postharvest storage. They will also be delivered back to our ARS lab that has expertise on comparative genomcis and functional analysis of fruit development, quality and ripening genes.