Molecular Ecology (2006) 15, 3045–3059 doi: 10.1111/j.1365-294X.2006.02981.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd Comparison of gene expression in segregating families identifies genes and genomic regions involved in a novel adaptation, zinc hyperaccumulation VICTOR FILATOV,* JOHN DOWDLE,* NICHOLAS SMIRNOFF,* BRIAN FORD-LLOYD,† H. JOHN NEWBURY † and MARK R. MACNAIR * *School of Biosciences, University of Exeter, Prince of Wales Rd, Exeter Devon EX4 4PS, †School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK Abstract One of the challenges of comparative genomics is to identify specific genetic changes associated with the evolution of a novel adaptation or trait. We need to be able to disassociate the genes involved with a particular character from all the other genetic changes that take place as lineages diverge. Here we show that by comparing the transcriptional profile of segregating families with that of parent species differing in a novel trait, it is possible to narrow down substantially the list of potential target genes. In addition, by assuming synteny with a related model organism for which the complete genome sequence is avail- able, it is possible to use the cosegregation of markers differing in transcription level to identify regions of the genome which probably contain quantitative trait loci (QTLs) for the character. This novel combination of genomics and classical genetics provides a very powerful tool to identify candidate genes. We use this methodology to investigate zinc hyperaccumulation in Arabidopsis halleri, the sister species to the model plant, Arabidopsis thaliana. We compare the transcriptional profile of A. halleri with that of its sister nonac- cumulator species, Arabidopsis petraea, and between accumulator and nonaccumulator F 3 s derived from the cross between the two species. We identify eight genes which consistently show greater expression in accumulator phenotypes in both roots and shoots, including two metal transporter genes (NRAMP3 and ZIP6), and cytoplasmic aconitase, a gene involved in iron homeostasis in mammals. We also show that there appear to be two QTLs for zinc accumulation, on chromosomes 3 and 7. Keywords: Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis petraea, cytoplasmic aconitase, hyper- accumulation, NRAMP3, ZIP6 Received 17 January 2006; revision accepted 27 March 2006 Introduction The advent of microarray technology has allowed valuable insights into the way in which sets of genes are involved in particular biological processes. The majority of experiments involve the comparison of the transcripts between different tissues or environmental conditions within a single species. In principle, it is possible to infer that the majority of differences observed in the transcriptome in such experiments are related to the biological process under study. However, in evolutionary genomics, we wish to compare how the transcriptome has changed alongside the evolution of a phenotypic character or genetic adaptation, and for this we need to compare the transcriptome of different species or ecotypes. In this case, the target transcriptional changes associated with the evolving or adapting trait will be buried within a mass of other differences resulting from unrelated evolutionary events that have occurred in parallel with the evolution of the character under investigation. However, using interspecies-crosses and derived segregating families, one can identify those transcriptional changes that are co-inherited with a target trait. In addition, if the species under investigation are closely related to a Correspondence: Mark Macnair, Fax: 01392 263700; E-mail: m.r.macnair@exeter.ac.uk