Ebrahimi (Purdue) - Reference Genomes

Seven Juglans spp. and seven F1 hybrids originated from the Midwestern United States, California, and Europe and an additional 26 J. regia samples obtained from the Indian Nut and Fruit Growers Association were used for growth and winterkill data collection. Winterkill damage was assessed by measuring for height and diameter at breast height for study trees. Field and growth data were analyzed using SAS software with PROC GLM mode and Fisher's least significant difference(LSD) test was applied when ANOVA were significant. After RNA extraction a qPCR template was generated from 1 µg total RNA used for First-strand cDNA synthesis with the Superscript III (Invitrogen, Carlsbad, USA) kit. Primers for qPCR were designed with Primer3 (http://frodo.wi.mit.edu/primer3/) using the J. regia genome sequence data available in the NCBI database (https://www.ncbi.nlm.nih.gov/). Actin was used as an internal qPCR control to measure gene expression. qPCR was performed using the Bio-RAD Real-time System (CFX connect, USA) with a SYBR Premix ExTaq Kit (Takara Bio, USA). Analyses of field and electrolyte leakage data were made on 12 Juglans samples and were sequenced with 100 bp paired-end reads at Purdue Genomics Center (https://www.purdue.edu/hla/sites/genomics/) using high throughput Illumina High seq 2500. Raw data were trimmed with Trimmomatic software (Bolger et al. 2014). Sequence fragments were assembled with SOAP-denovo software (Luo et al. 2012) using trimmed reads. Paired-end reads were mapped to the J. regia reference genome (Martínez-García et al. 2016) using the Burrow253 Wheeler Aligner (BWA), Picard-tools (Langmead et al. 2009; McKenna et al. 2010; Wysoker et al. 2013) and SNP calling with the Haplotype Caller tool from Genome Analysis Tool-kit (GATK) (McKenna et al. 2010). Consensus genome extracted with VCF tools and SAMtools. The assembled whole genome was used for whole gene prediction running AUGUSTUS (Stanke et al. 2006).