ABSTRACT: Grain yield and protein content were determined for six wheat cultivars grown over three years at multiple sites and at multiple N-fertilizer inputs. Although grain protein was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to calculate GPD across environment and to correlate gene expression in the developing caryopsis with this trait. The yield and protein content were initially adjusted for nitrogen fertilizer inputs, and then adjusted for yield (to remove the negative correlation) resulting in environmental corrected GPD. The transcriptome data for all samples were subjected to Principal Component Analysis (PCA) and ANOVA to identify individual Principal Components (PCs) correlating with GPD alone. Scores of the selected PCs significantly related to cultivar differences and GPD but not to the yield or protein content were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Sets of genes significant for these PCs and hence GPD were identified as candidate genes determining cultivar differences in GPD. Microarray profiling has been used to identify the links between gene expression and grain protein content in 6 different varietes of wheat grown at 2 different sites, 3 N levels and during 3 growth seasons. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted . In the present publication, data from two sites are included: Rothamsted and RAGTResearch (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted Research (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples.
