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International audience; All crop species are current or ancient polyploids. Following whole genome duplication, structural and functional modifications result in differential gene content or regulation in the duplicated regions, which can play a fundamental role in the diversification of genes underlying complex traits. We have investigated this issue in Brassica napus, a species with a highly duplicated genome, with the aim of studying the structural and functional organization of duplicated regions involved in quantitative resistance to stem canker, a disease caused by the fungal pathogen Leptosphaeria maculans. Genome-wide association analysis on two oilseed rape panels confirmed that duplicated regions of ancestral blocks E, J, R, U, and W were involved in resistance to stem canker. The structural analysis of the duplicated genomic regions showed a higher gene density on the A genome than on the C genome and a better collinearity between homoeologous regions than paralogous regions, as overall in the whole B. napus genome. The three ancestral sub-genomes were involved in the resistance to stem canker and the fractionation profile of the duplicated regions corresponded to what was expected from results on the B. napus progenitors. About 60% of the genes identified in these duplicated regions were single-copy genes while less than 5% were retained in all the duplicated copies of a given ancestral block. Genes retained in several copies were mainly involved in response to stress, signaling, or transcription regulation. Genes with resistance-associated markers were mainly retained in more than two copies. These results suggested that some genes underlying quantitative resistance to stem canker might be duplicated genes. Genes with a hydrolase activity that were retained in one copy or R-like genes might also account for resistance in some regions. Further analyses need to be conducted to indicate to what extent duplicated genes contribute to the expression of the resistance phenotype.

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

Background All sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing. Results This study provides a comprehensive analysis of the origins of lineage-specific genes (LSGs) in Arabidopsis thaliana that are restricted to the Brassicaceae family. In this study, lineage-specific genes within the nuclear (1761 genes) and mitochondrial (28 genes) genomes are identified. The evolutionary origins of two thirds of the lineage-specific genes within the Arabidopsis thaliana genome are also identified. Almost a quarter of lineage-specific genes originate from non-lineage-specific paralogs, while the origins of ~10% of lineage-specific genes are partly derived from DNA exapted from transposable elements (twice the proportion observed for non-lineage-specific genes). Lineage-specific genes are also enriched in genes that have overlapping CDS, which is consistent with such novel genes arising from overprinting. Over half of the subset of the 958 lineage-specific genes found only in Arabidopsis thaliana have alignments to intergenic regions in Arabidopsis lyrata, consistent with either de novo origination or differential gene loss and retention, with both evolutionary scenarios explaining the lineage-specific status of these genes. A smaller number of lineage-specific genes with an incomplete open reading frame across different Arabidopsis thaliana accessions are further identified as accession-specific genes, most likely of recent origin in Arabidopsis thaliana. Putative de novo origination for two of the Arabidopsis thaliana-only genes is identified via additional sequencing across accessions of Arabidopsis thaliana and closely related sister species lineages. We demonstrate that lineage-specific genes have high tissue specificity and low expression levels across multiple tissues and developmental stages. Finally, stress responsiveness is identified as a distinct feature of Brassicaceae-specific genes; where these LSGs are enriched for genes responsive to a wide range of abiotic stresses. Conclusion Improving our understanding of the origins of lineage-specific genes is key to gaining insights regarding how novel genes can arise and acquire functionality in different lineages. This study comprehensively identifies all of the Brassicaceae-specific genes in Arabidopsis thaliana and identifies how the majority of such lineage-specific genes have arisen. The analysis allows the relative importance (and prevalence) of different evolutionary routes to the genesis of novel ORFs within lineages to be assessed. Insights regarding the functional roles of lineage-specific genes are further advanced through identification of enrichment for stress responsiveness in lineage-specific genes, highlighting their likely importance for environmental adaptation strategies.

Vertebrates appear about 500 million years ago in the fossil record. This is only 25–50 million years after the great explosion of multicellular invertebrate body plans in the early Cambrian. On a geological time scale, this interval is a ‘blink of an eye’, suggesting that the evolution of regulatory genes is likely to be important in the origins of vertebrates. Here we present evidence for a role of steroid receptors in this process based on a phylogenetic analysis suggesting that receptors for androgens, glucocorticoids, mineralocorticoids, and progesterone evolved from an ancestral steroid receptor gene by two successive duplications over a brief time that could have coincided with the origins of vertebrates. Moreover, the duplications of these steroid receptors may be additional evidence for the two duplications on a genome-scale that have been proposed to be important in the evolution of vertebrates. The two successive duplications of steroid receptor genes and their subsequent sequence divergence leading to steroid-specific receptors that regulate growth, development, reproduction and homeostasis in vertebrates may have been one of the events important in vertebrate survival after the Cambrian during global extinctions that occurred about 440 and 370 million years ago.

The letter by Denis Shields [1] comments on our article on the molecular evolution of CXC chemokines and their receptors [2], in which we hypothesized that the chemokines originate from the central nervous system (CNS). Shield’s comments hinged mainly on the evolutionary analyses. Clearly, inferences drawn from phylogenetic analyses do have their limitations. In the initial stages of the analyses, we constructed phylogenetic trees using an outgroup for both receptor and ligands. Although in general outgroups are chosen arbitrarily, we constructed a CXC receptor (CXCR) tree with CC chemokine receptors (CCRs) as an outgroup. The topology did not change from what was presented, except for CXCR6, which was located in the CCR cluster, close to the starting point of cluster formation but with low bootstrap values. In general, the tips of a phylogenetic tree are well resolved, whereas the deeper nodes are poorly resolved. ‘Supertrees’, which in theory could combine different types of information, and not merely DNA or protein sequence information, might be an alternative for studying long evolutionary histories [3]. We adopted the philosophy of a ‘supertree’ approach by substantiating our hypothesis using not only phylogenetic analyses but also other information, such as protein function, several CXC ligands (CXCL) sharing the same receptor, the presence of the ELR (glutamic acid, leucine, arginine) motif, and chromosomal localizations. Shields also argues that the absence of CXCR4 in Ciona favours a primordial immunological function of the CXC family. However, in this respect, the presence of CXCR4 in jawless fish is more important. Kuroda et al. [4] sequenced 9312 cDNA clones from a lamprey (Petromyzon marinus). The serendipitous finding of only CXCR4 could be a testimony to its ancestral status. The lamprey CXCR4 receptors cluster, with a high bootstrap value, with all other CXCR4 found in jawed fish, amphibia, birds and mammals. In addition, the architecture and sophistication of the CNS of Ciona differ markedly compared with that found in other more advanced chordates [5]. Although using a small number of amino acids to construct a phylogeny can lead to incorrect topologies, this can be overcome by the bootstrap test [6], which we used extensively to identify statistically reliable orthologues. The use of complete sequences is preferred to detect true orthologues, although when only incomplete sequences are available, the phylogenetic analyses can be performed in two ways, using pair-wise deletion or complete deletion of missing information. We performed both, and the results were essentially identical topologies with only minor deviations, notably the position of the Xenopus CXCR3 and rainbow trout CXCR. Phylogenetic trees are a hypothetical model for the evolutionary history of a gene family. A gene conversion event might result in two paralogous genes clustering closer together in trees than in reality. Gene conversions occur relatively frequently; Shields [7] observed that 45% of a control group of clustered genes showed evidence of gene conversion. However, this should not preclude phylogenetic analyses; we should merely be aware of the possibility of gene conversion imposing a bias on the analyses. The gene conversion events between the clustered CXCR1 and CXCR2 sequences analysed by Shields [7] using human, rabbit and rat sequences probably occurred between ,90 and 110 million years ago [8]. The evolutionary scale on which these gene conversions could bias the phylogenetic tree is therefore limited to the clustering of CXCR1 and CXCR2 in mammals. We believe that putative gene conversion events do not interfere with our conclusions and hypotheses derived from phylogenetic analyses on a larger, vertebrate-wide scale that comprises more CXCRs. Shields also touches upon the controversy surrounding genome duplication. Some studies favour whole-genome duplications, whereas others point to accumulated duplication of blocks or single chromosomes. Most data supporting whole genome duplications are derived from analyses of either the major histocompatibility complex (MHC) [9] or the Hox [10] gene clusters. This issue is still unresolved. Following either mode of duplication, it is apparent that extensive reshuffling and loss of genes or gene regions has occurred. This obscures the reconstruction of events occurring during the ‘birth’ of genomes [11]. Our hypotheses do not refute either the whole-genome duplication or the block duplication hypothesis. Regardless of the pitfalls associated with phylogenetic analyses, this approach has the potential to unravel the evolutionary history of the CXCL/CXCR multigene families. Our opinion, based on a combination of these analyses and additional data, is an attempt to set these gene families in a compelling evolutionary perspective. Corresponding author: B.M. Lidy Verburg-van Kemenade (lidy.vankemenade@ wur.nl). Update TRENDS in Immunology Vol.24 No.7 July 2003 356