Stephan Greiner

Horizontal transfer of chloroplast genomes between plant species

The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.

Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes

Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers.

Tuning a ménage à trois: Co‐evolution and co‐adaptation of nuclear and organellar genomes in plants

Plastids and mitochondria arose through endosymbiotic acquisition of formerly free‐living bacteria. During more than a billion years of subsequent concerted evolution, the three genomes of plant cells have undergone dramatic structural changes to optimize the expression of the compartmentalized genetic material and to fine‐tune the communication between the nucleus and the organelles. The chimeric composition of many multiprotein complexes in plastids and mitochondria (one part of the subunits being nuclear encoded and another one being encoded in the organellar genome) provides a paradigm for co‐evolution at the cellular level. In this paper, we discuss the co‐evolution of nuclear and organellar genomes in the context of environmental adaptation in species and populations. We highlight emerging genetic model systems and new experimental approaches that are particularly suitable to elucidate the molecular basis of co‐adaptation processes and describe how nuclear‐cytoplasmic co‐evolution can cause genetic incompatibilities that contribute to the establishment of hybridization barriers, ultimately leading to the formation of new species.

Why are most organelle genomes transmitted maternally

Why the DNA‐containing organelles, chloroplasts, and mitochondria, are inherited maternally is a long standing and unsolved question. However, recent years have seen a paradigm shift, in that the absoluteness of uniparental inheritance is increasingly questioned. Here, we review the field and propose a unifying model for organelle inheritance. We argue that the predominance of the maternal mode is a result of higher mutational load in the paternal gamete. Uniparental inheritance evolved from relaxed organelle inheritance patterns because it avoids the spread of selfish cytoplasmic elements. However, on evolutionary timescales, uniparentally inherited organelles are susceptible to mutational meltdown (Mullers ratchet). To prevent this, fall‐back to relaxed inheritance patterns occurs, allowing low levels of sexual organelle recombination. Since sexual organelle recombination is insufficient to mitigate the effects of selfish cytoplasmic elements, various mechanisms for uniparental inheritance then evolve again independently. Organelle inheritance must therefore be seen as an evolutionary unstable trait, with a strong general bias to the uniparental, maternal, mode.

The role of plastids in plant speciation

Understanding the molecular basis of how new species arise is a central question and prime challenge in evolutionary biology and includes understanding how genomes diversify. Eukaryotic cells possess an integrated compartmentalized genetic system of endosymbiotic ancestry. The cellular subgenomes in nucleus, mitochondria and plastids communicate in a complex way and co‐evolve. The application of hybrid and cybrid technologies, most notably those involving interspecific exchanges of plastid and nuclear genomes, has uncovered a multitude of species‐specific nucleo‐organelle interactions. Such interactions can result in plastome–genome incompatibilities, which can phenotypically often be recognized as hybrid bleaching, hybrid variegation or disturbance of the sexual phase. The plastid genome, because of its relatively low number of genes, can serve as a valuable tool to investigate the origin of these incompatibilities. In this article, we review progress on understanding how plastome–genome co‐evolution contributes to speciation. We genetically classify incompatible phenotypes into four categories. We also summarize genetic, physiological and environmental influence and other possible selection forces acting on plastid–nuclear co‐evolution and compare taxa providing molecular access to the underlying loci. It appears that plastome–genome incompatibility can establish hybridization barriers, comparable to the Dobzhansky–Muller model of speciation processes. Evidence suggests that the plastid‐mediated hybridization barriers associated with hybrid bleaching primarily arise through modification of components in regulatory networks, rather than of complex, multisubunit structures themselves that are frequent targets.

The complete nucleotide sequences of the five genetically distinct plastid genomes of Oenothera, subsection Oenothera: I. Sequence evaluation and plastome evolution

The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome–genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163 365 bp (plastome IV) to 165 728 bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsection-specific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common ancestor.

Molecular Marker Systems for Oenothera Genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome–genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9·8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.

Recurrent Loss of Sex Is Associated with Accumulation of Deleterious Mutations in Oenothera

Sexual reproduction is nearly universal among eukaryotes. Theory predicts that the rarity of asexual eukaryotic species is in part caused by accumulation of deleterious mutations and heightened extinction risk associated with suppressed recombination and segregation in asexual species. We tested this prediction with a large data set of 62 transcriptomes from 29 species in the plant genus Oenothera, spanning ten independent transitions between sexual and a functionally asexual genetic system called permanent translocation heterozygosity. Illumina short-read sequencing and de novo transcript assembly yielded an average of 16.4 Mb of sequence per individual. Here, we show that functionally asexual species accumulate more deleterious mutations than sexual species using both population genomic and phylogenetic analysis. At an individual level, asexual species exhibited 1.8 × higher heterozygosity than sexual species. Within species, we detected a higher proportion of nonsynonymous polymorphism relative to synonymous variation within asexual compared with sexual species, indicating reduced efficacy of purifying selection. Asexual species also exhibited a greater proportion of transcripts with premature stop codons. The increased proportion of nonsynonymous mutations was also positively correlated with divergence time between sexual and asexual species, consistent with Mullers ratchet. Between species, we detected repeated increases in the ratio of nonsynonymous to synonymous divergence in asexual species compared with sexually reproducing sister taxa, indicating increased accumulation of deleterious mutations. These results confirm that an important advantage of sex is that it facilitates selection against deleterious alleles, which might help to explain the dearth of extant asexual species.

GeSeq - versatile and accurate annotation of organelle genomes

Abstract We have developed the web application GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) for the rapid and accurate annotation of organellar genome sequences, in particular chloroplast genomes. In contrast to existing tools, GeSeq combines batch processing with a fully customizable reference sequence selection of organellar genome records from NCBI and/or references uploaded by the user. For the annotation of chloroplast genomes, the application additionally provides an integrated database of manually curated reference sequences. GeSeq identifies genes or other feature-encoding regions by BLAT-based homology searches and additionally, by profile HMM searches for protein and rRNA coding genes and two de novo predictors for tRNA genes. These unique features enable the user to conveniently compare the annotations of different state-of-the-art methods, thus supporting high-quality annotations. The main output of GeSeq is a GenBank file that usually requires only little curation and is instantly visualized by OGDRAW. GeSeq also offers a variety of optional additional outputs that facilitate downstream analyzes, for example comparative genomic or phylogenetic studies.

The Complete Nucleotide Sequences of the 5 Genetically Distinct Plastid Genomes of Oenothera, Subsection Oenothera: II. A Microevolutionary View Using Bioinformatics and Formal Genetic Data

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus–organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I–V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome–genome combinations that do not occur naturally often display interspecific plastome–genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome–genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.