Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-016-19-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Jul 1, 2017
End Date: Jun 30, 2018
1) Perform RNA-Seq and small RNA (sRNA) analysis from almond shoots exhibiting BF and control trees. 2) Bioinformatics analysis of RNA-Seq and sRNA data to identify differentially expressed Prunus transcriptome and sRNAs in almonds exhibiting BF compared to control trees.
We will collect shoots from Nonpareil and Peerless almond trees exhibiting bud failure (BF) and from control NonPareil plants without BF. Several trees have already been identified (Fresnedo-Ramirez et al., 2017). Samples will be collected from three trees at four growth stages; flowering, spring shoot growth, nut filling, and new growth during late summer and fall. This will provide us 48 samples. Collected samples will be stored at -800C. Total RNA from frozen tissue will be isolated and polyA+ RNA will be prepared using oligo dT. RNA-Seq libraries from these samples will be prepared as described in Nagalakshmi et al., (2008 & 2010). For sRNA, low molecular weight RNA isolation and generation of sRNA libraries will be prepared by following the methods described in Chellappan et al., (2009). We will perform sequencing and analysis of 48 libraries prepared from polyA+ RNA and 48 libraries prepared from sRNA analyses, which would amount to a total of 96 cDNA libraries. We will map quality-filtered RNA-Seq and sRNA reads to coding-regions (ca. 27,852) and noncoding RNAs of peach (P. persica) genome (International Peach Genome Initiative et al., 2013) using the Burrows-Wheeler Aligner (BWA) and identify differentially expressed transcripts using Cufflinks (Trapnell et al., 2012). From this analysis, we will identify differentially regulated genes or sRNAs between BF trees compared to control. We will validate results of differentially expressed genes or sRNAs using real-time RT-qPCR. A University of California (UC), Davis, laboratory has extensive experience in RNA-Seq and sRNA analyses in tomato, maize, and Arabidopsis plants. A research scientist at the UCD Genome Center, is the lead scientist who developed RNA-Seq technology (Nagalakshmi et al., 2008) and will be provide expertise in RNA library preparations and next generation sequencing. The ARS scientist's lab has extensive experience using next generation sequencing to detect and characterize previously unknown viruses in woody plants. Using a similar approach, involving deep sequencing of cDNA libraries prepared from dsRNA, we recently found a new DNA virus called Grapevine red blotch-associated virus in wine grapes exhibiting reduced sugar accumulation and this virus is widely distributed in California and other major wine grape production areas in the U.S. and Canada (Al Rwahnih et al., 2013; Sudarshana et al., 2014). A new luteovirus has also been found in nectarine trees by this method (Bag et al., 2015).