cis and trans eQTLs Reveal the Location of Major Gene Expression Switches in Apple Rootstocks

Gene expression quantitative trait loci (eQTLs) can be obtained by mapping gene expression values in a segregating population.  One hundred and eighty-six F₁ progeny from the cross of apple rootstocks ‘Ottawa 3’× ‘Robusta 5’ (O3 x R5) were used for map construction and QTL analysis. Gene expression traits were measured on apical tissues (expanding leaves and stem) of a subset (48 individuals) of the O3R5 population through DNA microarray analysis with a second generation NimbleGen apple microarray containing 135,000 probes per plex and representing 26,017 unique apple transcripts.  Normalized gene expression data were treated as independent phenotypes for the purpose of QTL mapping with the MapQTL6 software.  A stepwise approach was used to identify eQTLs: preliminary eQTL analysis was performed by a Kruskal-Wallis test on all 26,017 features, subsequently, Interval Mapping (IM) was performed to identify putative eQTL regions.  The resulting 26,017 outputs were parsed for eQTLs having LOD values of 5.0 and higher and selected for further analysis.  The genetic location of all eQTL peaks with LOD 5.0 and higher was extracted from the MQ0 output files and combined with the physical location of the features mapped onto the Reference Apple Genome Sequence (V1).  The resulting file was used to plot eQTLs based on intensity (LOD), putative physical location and genetic location.  The plot revealed complex patterns of cis and trans eQTLs with “hot spots” in various genetic locations which might signify the pleiotropic effect of major transcription factors segregating in the progeny or other transcriptional switches (micro-RNAs, methylation, etc.).  Further annotation of the microarray features allowed the identification of hundreds of inherited patterns of gene expression belonging to know pathways and their metabolic products.  For example several known and unknown flavonoid compounds clustered with the expression profile of APPLE00R00024377 (At3g55120) a chalcone-flavanone isomerase located on Chr.12 whose expression segregated 1:1 in the O3R5 population.  There was also a significant correlation between expression of APPLE00R00024377 on the expression of the next pathway step APPLE0F000003679 annotated as a flavonoid 3' hydroxylase IIb [Malus x domestica] found on Chromosome 6, then APPLE0F000019828 (eQTL on LG07 at 36.958 cM with LOD of 9.13 physically located on Chr13 annotated as a putative UDP-galactose-flavonoid 3-O-galactosyltransferase [Malus x domestica]), and APPLE0F000021492 (eQTL located on LG07 at 36.958 cM with LOD of 14.04 physically located on Chr13 annotated as a UDP-glucose:flavonoid 7-O-glucosyltransferase [Malus x domestica]).  While this type of information may be useful to discover gene networks and major switches of gene networks, it is also useful to determine the complexity of a trait for breeding purposes and underscores the importance of the influence of the “genetic background” when transferring major factors during breeding.