Alessia Guggisberg

RAD in the realm of next‐generation sequencing technologies

The first North American RAD Sequencing and Genomics Symposium, sponsored by Floragenex (http://www.floragenex.com/radmeeting/), took place in Portland, Oregon (USA) on 19 April 2011. This symposium was convened to promote and discuss the use of restriction‐site‐associated DNA (RAD) sequencing technologies. RAD sequencing is one of several strategies recently developed to increase the power of data generated via short‐read sequencing technologies by reducing their complexity ( Baird et al. 2008 ; Huang et al. 2009 ; Andolfatto et al. 2011 ; Elshire et al. 2011 ). RAD sequencing, as a form of genotyping by sequencing, has been effectively applied in genetic mapping and quantitative trait loci (QTL) analyses in a range of organisms including nonmodel, genetically highly heterogeneous organisms ( Table 1 ; Baird et al. 2008 ; Baxter et al. 2011 ; Chutimanitsakun et al. 2011 ; Pfender et al. 2011 ). RAD sequencing has recently found applications in phylogeography ( Emerson et al. 2010 ) and population genomics ( Hohenlohe et al. 2010 ). Considering the diversity of talks presented during this meeting, more developments are to be expected in the very near future.

Genomics of Compositae weeds: EST libraries, microarrays, and evidence of introgression.

PREMISE OF STUDY Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we describe the development of genomic tools and resources for 11 weeds from the Compositae family that will serve as a basis for subsequent population and comparative genomic analyses. Because hybridization has been suggested as a stimulus for the evolution of invasiveness, we also analyze these genomic data for evidence of hybridization. METHODS We generated 22 expressed sequence tag (EST) libraries for the 11 targeted weeds using Sanger, 454, and Illumina sequencing, compared the coverage and quality of sequence assemblies, and developed NimbleGen microarrays for expression analyses in five taxa. When possible, we also compared the distributions of Ks values between orthologs of congeneric taxa to detect and quantify hybridization and introgression. RESULTS Gene discovery was enhanced by sequencing from multiple tissues, normalization of cDNA libraries, and especially greater sequencing depth. However, assemblies from short sequence reads sometimes failed to resolve close paralogs. Substantial introgression was detected in Centaurea and Helianthus, but not in Ambrosia and Lactuca. CONCLUSIONS Transcriptome sequencing using next-generation platforms has greatly reduced the cost of genomic studies of nonmodel organisms, and the ESTs and microarrays reported here will accelerate evolutionary and molecular investigations of Compositae weeds. Our study also shows how ortholog comparisons can be used to approximately estimate the genome-wide extent of introgression and to identify genes that have been exchanged between hybridizing taxa.

Sequencing of the genus Arabidopsis identifies a complex history of nonbifurcating speciation and abundant trans-specific polymorphism

The notion of species as reproductively isolated units related through a bifurcating tree implies that gene trees should generally agree with the species tree and that sister taxa should not share polymorphisms unless they diverged recently and should be equally closely related to outgroups. It is now possible to evaluate this model systematically. We sequenced multiple individuals from 27 described taxa representing the entire Arabidopsis genus. Cluster analysis identified seven groups, corresponding to described species that capture the structure of the genus. However, at the level of gene trees, only the separation of Arabidopsis thaliana from the remaining species was universally supported, and, overall, the amount of shared polymorphism demonstrated that reproductive isolation was considerably more recent than the estimated divergence times. We uncovered multiple cases of past gene flow that contradict a bifurcating species tree. Finally, we showed that the pattern of divergence differs between gene ontologies, suggesting a role for selection.

Disentangling Reticulate Evolution in an Arctic–Alpine Polyploid Complex

Although polyploidy plays a fundamental role in plant evolution, the elucidation of polyploid origins is fraught with methodological challenges. For example, allopolyploid species may confound phylogenetic reconstruction because commonly used methods are designed to trace divergent, rather than reticulate patterns. Recently developed techniques of phylogenetic network estimation allow for a more effective identification of incongruence among trees. However, incongruence can also be caused by incomplete lineage sorting, paralogy, concerted evolution, and recombination. Thus, initial hypotheses of hybridization need to be examined via additional sources of evidence, including the partitioning of infraspecific genetic polymorphisms, morphological characteristics, chromosome numbers, crossing experiments, and distributional patterns. Primula sect. Aleuritia subsect. Aleuritia (Aleuritia) represents an ideal case study to examine reticulation because specific hypotheses have been derived from morphology, karyology, interfertility, and distribution to explain the observed variation of ploidy levels, ranging from diploidy to 14-ploidy. Sequences from 5 chloroplast and 1 nuclear ribosomal DNA (nrDNA) markers were analyzed to generate the respective phylogenies and consensus networks. Furthermore, extensive cloning of the nrDNA marker allowed for the identification of shared nucleotides at polymorphic sites, investigation of infraspecific genetic polymorphisms via principal coordinate analyses PCoAs, and detection of recombination between putative progenitor sequences. The results suggest that most surveyed polyploids originated via hybridization and that 2 taxonomic species formed recurrently from different progenitors, findings that are congruent with the expectations of speciation via secondary contact. Overall, the study highlights the importance of using multiple experimental and analytical approaches to disentangle complex patterns of reticulation.

Allopolyploid Origin of the Mediterranean Endemic, Centaurium bianoris (Gentianaceae), Inferred by Molecular Markers

Abstract Centaurium bianoris (Gentianaceae) is restricted to Majorca, the main island of the Balearic Archipelago. This tetraploid species is characterised by salmon-coloured corollas (var. bianoris), but pink (var. roseum) and yellow (var. sulfureum) varieties have also been described. An allopolyploid origin has been proposed between the diploids C. maritimum (yellow flowers) and C. tenuiflorum var. acutiflorum (pink flowers), both occurring on Majorca and in other places of the Mediterranean basin. In this study, we tested the proposed hybrid origin of C. bianoris by using RAPD fingerprinting, and both direct and cloned sequences of the nuclear ribosomal ITS, and the chloroplast trnLF regions. Our molecular data confirmed the hypothesis of an allotetraploid origin of C. bianoris via hybridisation between C. tenuiflorum and C. maritimum, the latter being the maternal parent. The so-called varieties roseum and sulfureum appeared to be only floral morphs that may have arisen via genomic processes such as gene silencing. Hybridisation is probably the cause of the ITS sequence polymorphism observed in C. bianoris, whereas backcrosses with either parent may be responsible for the apparent bidirectional homogenisation observed in ITS clones. Finally, the polyphyletic behaviour of C. bianoris on the ITS cladogram, combined with the differential rates of homogenisation observed in ITS sequences, may denote a recurrent origin for that taxon. This result contrasts with the narrow distribution of C. bianoris, compared to that of its diploid parents, suggesting instead a single origin for this hybrid.

Genomic Origin and Organization of the Allopolyploid Primula egaliksensis Investigated by in situ Hybridization

BACKGROUND AND AIMS Earlier studies have suggested that the tetraploid Primula egaliksensis (2n = 40) originated from hybridization between the diploids P. mistassinica (2n = 18) and P. nutans (2n = 22), which were hypothesized to be the maternal and paternal parent, respectively. The present paper is aimed at verifying the hybrid nature of P. egaliksensis using cytogenetic tools, and to investigate the extent to which the parental genomes have undergone genomic reorganization. METHODS Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with ribosomal DNA (rDNA) probes, together with sequencing of the internal transcribed spacer (ITS) region of the rDNA, were used to identify the origin of P. egaliksensis and to explore its genomic organization, particularly at rDNA loci. KEY RESULTS GISH showed that P. egaliksensis inherited all chromosomes from P. mistassinica and P. nutans and did not reveal major intergenomic rearrangements between the parental genomes (e.g. interchromosomal translocations). However, karyological comparisons and FISH experiments suggested small-scale rearrangements, particularly at rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromatic knobs characteristic of the maternal parent P. mistassinica and maintained only the rDNA loci of P. nutans. These results corroborated sequence data indicating that most ITS sequences of P. egaliksensis were of the paternal repeat type. CONCLUSIONS The lack of major rearrangements may be a consequence of the considerable genetic divergence between the putative parents, while the rapid elimination of the ITS repeats from the maternal progenitor may be explained by the subterminal location of ITS loci or a potential role of nucleolar dominance in chromosome stabilization. These small-scale rearrangements may be indicative of genome diploidization, but further investigations are needed to confirm this assumption.

Transcriptome divergence between introduced and native populations of Canada thistle, Cirsium arvense

Introduced plants may quickly evolve new adaptive traits upon their introduction. Canada thistle (Cirsium arvense - Cardueae, Asteraceae) is one of the worst invasive weeds worldwide. The goal of this study is to compare gene expression profiles of native (European) and introduced (North American) populations of this species, to elucidate the genetic mechanisms that may underlie such rapid adaptation. We explored the transcriptome of ten populations (five per range) of C. arvense in response to three treatments (control, nutrient deficiency and shading) using a customized microarray chip containing 63 690 expressed sequence tags (ESTs), and verified the expression level of 13 loci through real-time quantitative PCR. Only 2116 ESTs (3.5%) were found to be differentially expressed between the ranges, and 4458 ESTs (7.1%) exhibited a significant treatment-by-range effect. Among them was an overrepresentation of loci involved in stimulus and stress responses. Cirsium arvense has evolved different life history strategies on each continent. The two ranges notably differ with regard to R-protein mediated defence, sensitivity to abiotic stresses, and developmental timing. The fact that genotypes from the Midwest exhibit different expression kinetics than remaining North American samples further corroborates the hypothesis that the New World has been colonized twice, independently.

Exogenous selection rather than cytonuclear incompatibilities shapes asymmetrical fitness of reciprocal Arabidopsis hybrids.

Reciprocal crosses between species often display an asymmetry in the fitness of F1 hybrids. This pattern, referred to as isolation asymmetry or Darwins corollary to Haldanes rule, is a general feature of reproductive isolation in plants, yet factors determining its magnitude and direction remain unclear. We evaluated reciprocal species crosses between two naturally hybridizing diploid species of Arabidopsis to assess the degree of isolation asymmetry at different postmating life stages. We found that pollen from Arabidopsis arenosa will usually fertilize ovules from Arabidopsis lyrata; the reverse receptivity being less complete. Maternal A. lyrata parents set more F1 hybrid seed, but germinate at lower frequency, reversing the asymmetry. As predicted by theory, A. lyrata (the maternal parent with lower seed viability in crosses) exhibited accelerated chloroplast evolution, indicating that cytonuclear incompatibilities may play a role in reproductive isolation. However, this direction of asymmetrical reproductive isolation is not replicated in natural suture zones, where delayed hybrid breakdown of fertility at later developmental stages, or later-acting selection against A. arenosa maternal hybrids (unrelated to hybrid fertility, e.g., substrate adaptation) may be responsible for an excess of A. lyrata maternal hybrids. Exogenous selection rather than cytonuclear incompatibilities thus shapes the asymmetrical postmating isolation in nature.