## 7 Building a Molecular Toolbox for Engineering of Targeted Disease Resistance  
### Materials and Methods for transcriptomics of Pratylenchus-Challenged Wheat Roots  
_Pratylenchus neglectus_ and _P. thornei_, obtained from soil samples, were kind gifts from Drs. [PERSON] and [PERSON], Oregon State University, Pendleton, Oregon, USA. Nematodes were cultured on sterilized carrot disks according to [PERSON] et al. [106]. Seeds of susceptible spring wheat cultivar Scarlet [107] were sown singly in 6-inch plastic cones (Stuewe and Sons, Corvallis, OR, USA) containing autoclaved sand. Seedlings were grown at 25 \({}^{\circ}\)C, 16h lt/20 \({}^{\circ}\)C, 8 h dk. Nematodes were introduced to roots of 14-day-old plants as soil drenches of 2000 mixed-stage (J2-J4) juveniles. Each experiment consisted of four treatments, three plants per treatment: (1) _Pratylenchus neglectus_ only (Pn); (2) _P. thornei_ only (Pt); (3) a 1:1 mixture of _P. neglectus_ and _P. thornei_ (PnPt); and (4) no-hematode control (Cont). After a 21-day treatment period, roots were rinsed to remove sand, blotted gently, and pooled by treatment into tubes containing liquid nitrogen. Harvested roots were stored at \(-80\)\({}^{\circ}\)C prior to RNA isolation. The experiment was repeated twice for a total of three biological replications and 12 RNA samples.  
Total RNA was obtained from treated roots using a modified TRIzol method [83]. RNA samples were processed and sequenced by MRDNA, Shallowwater, TX, USA. RNA concentration was determined using the Qubi\({}^{\circledR}\) RNA Assay Kit (Life Technologies, Carlsbad, CA, USA), and information for RNA quality and average library size was obtained using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA quality was based on sizes of all RNA fragments obtained using the Bioanalyzer and the Agilent 2100 software [108]. Libraries of each of the 12 RNA samples were prepared from 1.0 \(\upmu\)g of total RNA using the TruSeq\({}^{\circledR}\) RNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Libraries were indexed and prepared for polymerase chain reaction amplification using barcodes and adaptors. A total of 2 nM of each library were pooled; 5 pM of the pooled mix was subjected to Illumina HiSeq 2500 paired-end \(2\times 150\) sequencing.  
Raw sequence reads were filtered for quality, and adaptors, index barcodes, and ambiguous bases were removed using the CLC Genomics Workbench Trim Sequences tools (Qiagen Bioinformatics, Redwood City, CA, USA). De novo assembly of filtered reads was conducted using the [PERSON] graphing algorithm [109]. Paired-end reads exhibiting overlapping sequences were merged and reads greater than 85 bp (20-22 bp overlap) were assembled into contiguous sequences (contigs) representing the expressed transcripts. Expressed transcript groups were mapped to the _Triticum aestivum_ available at the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0 coding sequence database [110]. The IWGSC database represented putative coding sequences on the long and short arms of each of the seven chromosomes of the A, B, and D genomes of hexaploid wheat. Transcript groups also were annotated using Blast2 GO (BioBam Bioinformatics S.L., Valencia, Spain), which provided initial sequence and gene function information. Numbers of transcripts among samples were normalized using reads per kilobase of transcript per million mapped reads (RPKM) [111] so that transcript abundance between treatments could be compared. Transcript groups having an RKPM value \(\geq\)50 for at least one of four treatments (Pn, Pt, Pn and Pt, or control) were examined further to identify those that were induced in the presence of nematodes. Transcript groups that showed a Pn/Cont, Pt/Cont, and/or PnPt/Cont RPKM ratio of \(>\)2.0 were considered to be induced during Pratylenchus challenge. The arbitrary cutoff of RPKM \(\geq\)50 was intended to rise above \"background\" expression and increase the identity of causative loci.