Verticillium dahliae disease resistance and the regulatory pathway for maturity and tuberization in potato

Purpose: Verticillium dahliae is a fungal plant pathogen. The goals of the study are to examine gene networks involved in controlling resistance to V. dahliae in potato using expression quantitative trait loci (eQTL) mapping. Methods: 91 clones from a diploid potato mapping population 15143 and the parental lines 07506-01 and 12120-03 from the Agriculture and Agri-Food Canada in Fredericton, New Brunswick, Canada were propagated in a V. dahliae-infested field in triplicate plots of five plants in a randomized complete block design. Tetraploid varieties Shepody, Hindenburg, Kennebec, Russet Burbank, Atlantic and Green Mountain were planted similarly and included in the analysis. Green Mountain was also planted in additional plots and used a guard row clone. Leaf samples were taken at 70 days after planting. Two µg of total RNA per sample was used to construct DeepSAGE tag libraries using a modification to facilitate direct sequencing of the amplicons by Illumina sequencing. The samples from 91 clones were bar-coded with a different identification key. Samples were diluted to a final concentration of 10 nM and were pooled. Each of the three biological replicates was done in a separate pool. Final pool concentrations were estimated using Quant-iT-PicoGreen prior to template DNA hybridization and sequencing on an Illumina Genome Analyzer II according to the manufacturer's instructions. Results: Tag count data were transformed to the relative unit of counts per million. Tags included in the analysis had to be present in at least one biological replicate. Clonal variation between samples was detected (p-value ≤ 0.05) using the exact test for a negative binomial distribution . Tags that did not have significant clonal variation were removed, leaving 6248 tags. The LS mean of tag count was calculated for each tag for each clone. The 21-nt tag sequences were aligned against PGSC S. tuberosum group Phureja DM1-3 transcripts (v3.4) using blastn. Hits with E-value ≤ 0.5 were filtered in. Where there were multiple blast hits meeting E-value criteria, only the one with the longest alignment was included. 4198 tags were assigned single-hit annotations in this manner and were used for eQTL analysis. Conclusions: The results indicated that for most genes the eQTL controlling expression was mapped near the gene (cis-eQTL). There were also many trans-eQTLs, where were eQTLs for a gene that were found on another chromosome particularly on chromosome 5. Three sequencing runs were done in 8 channels. RunA was a barcoded pool of all clones from block A/replicate 1. RunB was a barcoded pool of all clones from block B/replicate 2. RunC eas a barcoded pool of all clones from block C/replicate3