Evolutionary transgenomics: prospects and challenges

Abstract

Many advances in our understanding of the genetic basis of species differences have arisen from transformation experiments, which allow us to study the effect of genes from one species (the donor) when placed in the genetic background of another species (the recipient). Such interspecies transformation experiments are usually focused on candidate genes - genes that, based on work in model systems, are suspected to be responsible for certain phenotypic differences between the donor and recipient species. We suggest that the high efficiency of transformation in a few plant species, most notably Arabidopsis thaliana, combined with the small size of typical plant genes and their cis-regulatory regions allow implementation of a screening strategy that does not depend upon a priori candidate gene identification. This approach, transgenomics, entails moving many large genomic inserts of a donor species into the wild type background of a recipient species and then screening for dominant phenotypic effects. As a proof of concept, we recently conducted a transgenomic screen that analyzed more than 1100 random, large genomic inserts of the Alabama gladecress Leavenworthia alabamica for dominant phenotypic effects in the A. thaliana background. This screen identified one insert that shortens fruit and decreases A. thaliana fertility. In this paper we discuss the principles of transgenomic screens and suggest methods to help minimize the frequencies of false positive and false negative results. We argue that, because transgenomics avoids committing in advance to candidate genes it has the potential to help us identify truly novel genes or cryptic functions of known genes. Given the valuable knowledge that is likely to be gained, we believe the time is ripe for the plant evolutionary community to invest in transgenomic screens, at least in the mustard family Brassicaceae where many species are amenable to efficient transformation.

Keywords: developmental system drift; evo-devo; evolution; genetic screens; speciation genes; transformation; transgenomics.

FIGURE 1

A possible mechanism of development system drift (DSD). DSD occurs when two lineages remain phenotypically unchanged but undergo genetic divergence (True and Haag, 2001). The phenomenon depends primarily on evolutionary changes that influence the way that proteins interact with each other and with DNA sequences (Haag, 2007; Landry et al., 2007), although changes in miRNA’s and their targets (Mallory et al., 2004) have a potential role as well. To understand how such phenomena can lead to a dominant phenotype in a transgenomic screen, consider a hypothetical example in which two proteins, A and B, dimerize but need to dissociate during normal development. In a hypothetical ancestor, pockets in both proteins destabilize the dimer enough to permit dissociation. In the lineage leading to species 1, protein A (A1) loses its pocket, but dissociation is still achieved thanks to the pocket in protein B1. Conversely, the pocket in protein B2 has been lost on the lineage leading to species 2. Moving protein A1 in species 2 (or B2 into species 1) will cause formation of a non-dissociable dimer, resulting in a dominant disruption of normal development. When development is disrupted sufficiently to cause inviability or sterility, DSD can enforce reproductive isolation between lineages, because hybrids do not survive to reproduce. In that case the pattern conforms to the Dobzhansky–Muller model of speciation [e.g., (Bomblies et al., 2007; Landry et al., 2007)], showing that transgenomics offers a novel way to identify genetic interactions that could contribute to speciation.

FIGURE 2

Dominant phenotypic effects found in a transgenomic screen could reflect phenotypic divergence or developmental system drift between donor and recipient species. In a transgenomic screen random genomic fragments from a donor species are introduced by transformation into a recipient species. The transformants are screened for phenotypes that differ from the recipient species. When an insert causes a phenotype of the donor species to be found, for example round, flattened fruits (plant A), it is likely that the transgene contains a major gene contributing to the evolution of that phenotype. If the phenotype is intermediate between the donor and recipient species, for example pinnately lobed (plant B) rather than either entire or pinnately compound leaves, then the transgene is a candidate for being one of several genes that changed during phenotypic divergence. If the phenotype resembles neither the donor nor the recipient species, as for example in having five rather than four petals (plant C), then developmental system drift is suggested.

FIGURE 3

The proportion of major genes expected to be uncovered in a transgenomic screen. Based on evolutionary principles we would predict that 50% of the major genes responsible for a phenotypic difference between the donor and recipient species will be found in a unidirectional transgenomic screen. To see why this is so, imagine a potential donor and recipient species for a transgenomic screen that differ in a phenotype (circle vs. square, respectively). Without further information it is equally likely that: (A) the donor species has the derived phenotype, with a change having occurred on the lineage from the common ancestor to the donor species (upper panels), or (B) the donor has the ancestral phenotype, with a change having occurred on the lineage from the common ancestor to the recipient species (lower panels). The mutation that gave rise to the derived phenotype could have been fully recessive or at least partially dominant. If the donor has a derived phenotype that is dominant (top right), or it has an ancestral phenotype that is dominant (bottom right), then moving the causal gene into the recipient will yield a dominant phenotype. In approximately 50% of cases (left panels) the causal gene will not yield a dominant phenotype when moved from the donor to the recipient. The major genes missed in a unidirectional transgenomic screen could theoretically be found with a reciprocal screen in which the genome of the former recipient species is screened in the background of the former donor species.

FIGURE 4

Donor species genes can be screened for phenotypic effects in the recipient species by shotgun or clone-by-clone strategies. In a shotgun screen (left half of flow-chart) the library is transferred en masse into recipient plants, which are screened phenotypically. In a clone-by-clone screen (right half of flow-chart), clones are arranged in microtiter plates and transformants are generated for each isolated clone.