In order to begin characterizing the genetic basis of functional trait variation in the HSA, we are constructing QTL mapping and conducting analyses in a large Dubautia reticulata x Dubautia menziesii F2 family. The primary QTL analysis will examine the extent to which the suite of traits differentiating these two species, as identified in a different part of this project (functional trait analyses), are largely governed by pleiotropic effects of individual loci resulting in concurrent divergence in multiple traits

Several Dubautia menziesii and Dubautia reticulata plants currently growing at RSABG have been crossed to generate several sets of unrelated F1 families, and the seeds are currently being germinated. Reciprocal crosses between unrelated F1 individuals will be used to generate an outcross F2 family of 600-1000 seedlings. An outcross F2 design will be used because most Dubautia spp. are self-incompatible (Carr et al. 1986). A genetic linkage map will be constructed from ~100 individuals in the F2 family using the gene-based markers (developed in phase 3) supplemented with ~200-400 AFLP markers to ensure genome saturation and complete resolution of linkage groups. Markers from the genetic map will be oriented with the BAC contigs, providing a comprehensive physical map of the Dubautia genome. A physical map will facilitate identification and testing of positional candidate genes from subsequent QTL mapping and will facilitate linkage disequilibrium mapping in comparison to estimated empirical null distributions.

QTL mapping will be done in the F2 population, with a population size of ~600 F2 individuals, or ~300 individuals from each reciprocal cross.

We will utilize an integrated system of shotgun proteomics. Transcriptome profiles will be carefully compared to proteome profiles. Protein extracts from developing shoot and leaf tissue will be bulked by the two contrasting homozygous genotype classes in QTL regions for selected QTLs with broad relevance to functional trait divergence.