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Microalgal contamination in algal culture is a serious problem hampering the cultivation process, which can result in considerable economic and time losses. With the field of microalgal biotechnology on the rise, development of new tools for monitoring the cultures is of high importance. Here we present a case study of the detection of fast-growing green algae Chlorella vulgaris (as contaminant) in a diatom Phaeodactylum tricornutum culture using various approaches. We prepared mixed cultures of C. vulgaris and P. tricornutum in different cell-to-cell ratios in the range from 1:103 to 1:107. We compared the sensitivity among microscopy, cultivation-based technique, PCR, and qPCR. The detection of C. vulgaris contamination using light microscopy failed in samples containing cell ratios

The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

Dioecious plants possess diverse sex determination systems and unique mechanisms of reproductive organ development; however, little is known about how sex-linked genes shape the expression of regulatory cascades that lead to developmental differences between sexes. InSilene latifolia, a dioecious plant with stable dimorphism in floral traits, early experiments suggested that female-regulator genes act on the factors that determine the boundaries of the flower whorls. To identify these regulators, we sequenced the transcriptome of male flowers with fully developed gynoecia induced by rapid demethylation in the parental generation. As the hermaphrodite flower trait is holandric (transmitted only from male to male, inherited on the Y chromosome), we screened for genes that are differentially expressed between male, female, and hermaphrodite flowers. Dozens of candidate genes that are upregulated in hermaphrodite flowers compared to male and female flowers were detected and found to have putative roles in floral organization, affecting the expression of floral MADS-box and other genes. Amongst these genes, eight candidates were found to promote gynoecium formation in female and hermaphrodite flowers, affecting organ size, whorl boundary, and the expression of mainly B class flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using a field emission environmental scanning electron microscope. Our results reveal the principal regulatory pathways involved in sex-specific flower development in the classical model of dioecy,S. latifolia.

Intra-specific variability is a cornerstone of evolutionary success of species. Acquiring genetic material from distant sources is an important adaptive mechanism in bacteria, but it can also play a role in eukaryotes. In this paper, we investigate the nature and evolution of a chromosomal segment of panicoid (Poaceae, Panicoideae) origin occurring in the nuclear genomes of species of the barley genus Hordeum (Pooideae). The segment, spanning over 440 kb in the Asian Hordeum bogdanii and 219 kb in the South American Hordeum pubiflorum, resides on a pair of nucleolar organizer region (NOR)-bearing chromosomes. Conserved synteny and micro-collinearity of the segment in both species indicate a common origin of the segment, which was acquired before the split of the respective barley lineages 5-1.7 million years ago. A major part of the foreign DNA consists of several approximately 68 kb long repeated blocks containing five stress-related protein-coding genes and transposable elements (TEs). Whereas outside these repeats, the locus was invaded by multiple TEs from the host genome, the repeated blocks are rather intact and appear to be preserved. The protein-coding genes remained partly functional, as indicated by conserved reading frames, a low amount of non-synonymous mutations, and expression of mRNA. A screen across Hordeum species targeting the panicoid protein-coding genes revealed the presence of the genes in all species of the section Stenostachys. In summary, our study shows that grass genomes can contain large genomic segments obtained from distantly related species. These segments usually remain undetected, but they may play an important role in the evolution and adaptation of species.

Background and Aims Dioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY1Y2 sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes. Methods We flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons. Key Results We identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes. Conclusions The X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.

Background The evolution of dioecious plants is occasionally accompanied by the establishment of sex chromosomes: both XY and ZW systems have been found in plants. Structural studies of sex chromosomes are now being followed up by functional studies that are gradually shedding light on the specific genetic and epigenetic processes that shape the development of separate sexes in plants. Scope This review describes sex determination diversity in plants and the genetic background of dioecy, summarizes recent progress in the investigation of both classical and emerging model dioecious plants and discusses novel findings. The advantages of interspecies hybrids in studies focused on sex determination and the role of epigenetic processes in sexual development are also overviewed. Conclusions We integrate the genic, genomic and epigenetic levels of sex determination and stress the impact of sex chromosome evolution on structural and functional aspects of plant sexual development. We also discuss the impact of dioecy and sex chromosomes on genome structure and expression.

Background The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. Methods In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. Results We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. Conclusions Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

Background Polycomb repressive complexes 1 and 2 play important roles in epigenetic gene regulation by posttranslationally modifying specific histone residues. Polycomb repressive complex 2 is responsible for the trimethylation of lysine 27 on histone H3; Polycomb repressive complex 1 catalyzes the monoubiquitination of histone H2A at lysine 119. Both complexes have been thoroughly studied in Arabidopsis, but the evolution of polycomb group gene families in monocots, particularly those with complex allopolyploid origins, is unknown. Results Here, we present the in silico identification of the Polycomb repressive complex 1 and 2 (PRC2, PRC1) subunits in allohexaploid bread wheat, the reconstruction of their evolutionary history and a transcriptional analysis over a series of 33 developmental stages. We identified four main subunits of PRC2 [E(z), Su(z), FIE and MSI] and three main subunits of PRC1 (Pc, Psc and Sce) and determined their chromosomal locations. We found that most of the genes coding for subunit proteins are present as paralogs in bread wheat. Using bread wheat RNA-seq data from different tissues and developmental stages throughout plant ontogenesis revealed variable transcriptional activity for individual paralogs. Phylogenetic analysis showed a high level of protein conservation among temperate cereals. Conclusions The identification and chromosomal location of the Polycomb repressive complex 1 and 2 core components in bread wheat may enable a deeper understanding of developmental processes, including vernalization, in commonly grown winter wheat.

In the present study, we used Illumina sequencing technology (HiSeq 2000) to sequence the transcriptome of barley (Hordeum vulgare L., cv. Morex) under three different heavy metal stress conditions: copper, zinc and cadmium. For each of those metals, the concentration causing a 50% inhibitory effect for root growth (EC50) was determined. We sequenced the total RNA of both roots and shoots from barley with and without heavy metal treatments in three replicates. Raw reads of the transcriptome project have been deposited in NCBI's BioProject accession number PRJNA382490. The obtained transcriptomic data will be useful for further studies focusing on heavy metal tolerance and comparative transcriptome analysis in barley.

This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (http://dx.doi.org/10.24072/pci.evolbiol.100044).Sex chromosomes have repeatedly evolved from a pair of autosomes1. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced Y gene expression and eventual Y gene loss. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2–4. However, the early steps in the evolution of dosage compensation remain unknown and dosage compensation is poorly understood in plants5. Here we show a novel dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno for the evolution of X inactivation in mammals.

Switches in heterogamety are known to occur in both animals and plants. Although plant sex determination systems probably often evolved more recently than those in several well-studied animals, including mammals, and have had less time for switches to occur, we previously detected a switch in heterogamety in the plant genus Silene: section Otites has both female and male heterogamety, whereas S. latifolia and its close relatives, in a different section of the genus, Melandrium (subgenus Behenantha), all have male heterogamety. Here we analyse the evolution of sex chromosomes in section Otites, which is estimated to have evolved only about 0.55 MYA. Our study confirms female heterogamety in S. otites and newly reveals female heterogamety in S. borysthenica. Sequence analyses and genetic mapping show that the sex-linked regions of these two species are the same, but the region in S. colpophylla, a close relative with male heterogamety, is different. The sex chromosome pairs of S. colpophylla and S. otites each correspond to an autosome of the other species, and both differ from the XY pair in S. latifolia. Silene section Otites species are suitable for detailed studies of the events involved in such changes, and our phylogenetic analysis suggests a possible change from female to male heterogamety within this section. Our analyses suggest a possibility that has so far not been considered, change in heterogamety through hybridization, in which a male-determining chromosome from one species is introgressed into another one, and over-rides its previous sex-determining system.

Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.

Summary Silene vulgaris possesses ecotype-specific tolerance to high levels of copper in the soil. Although this was reported a few decades ago, little is known about this trait on a molecular level. The aim of this study was to analyze the transcription response to elevated copper concentrations in two S. vulgaris ecotypes originating from copper-contrasting soil types – copper-tolerant Lubietova and copper-sensitive Stranska skala. To reveal if plants are transcriptionally affected, we first analyzed the HMA7 gene, a known key player in copper metabolism. Based on BAC library screening, we identified a BAC clone containing a SvHMA7 sequence with all the structural properties specific for plant copper-transporting ATPases. The functionality of the gene was tested using heterologous complementation in yeast mutants. Analyses of SvHMA7 transcription patterns showed that both ecotypes studied up-regulated SvHMA7 transcription after the copper treatment. Our data are supported by analysis of appropriate reference genes based on RNA-Seq databases. To identify genes specifically involved in copper response in the studied ecotypes, we analyzed transcription profiles of genes coding Cu-transporting proteins and genes involved in the prevention of copper-induced oxidative stress in both ecotypes. Our data show that three genes (APx, POD and COPT5) differ in their transcription pattern between the ecotypes with constitutively increased transcription in Lubietova. Taken together, we have identified transcription differences between metallifferous and non-metalliferous ecotypes of S. vulgaris, and we have suggested candidate genes participating in metal tolerance in this species.

Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance.

Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed.

Silene latifolia (or white campion) possesses a well-established sex determination system with a dominant Y chromosome in males (the mammalian type). The heteromorphic sex chromosomes X and Y in S. latifolia largely stopped recombination; thus, we can expect a gradual genetic degeneration of the Y chromosome. It is well proven that neither diploid nor polyploid S. latifolia sporophytes can survive without at least one X, so the only life stage possessing the Y as the sole sex chromosome is the male gametophyte (pollen tube), while the female gametophyte seems to be X-dependent. Previous studies on anther-derived plants of this species showed that the obtained plants (largely haploid or dihaploid) were phenotypically and cytologically female. In this paper, we provide molecular evidence for the inviability of plants lacking the X chromosome. Using sex-specific PCR primers, we show that all plantlets and plants derived from anther cultures are female. In studying anther-derived diploid females by sequencing of X-linked markers, we demonstrate that these plants are really homozygous dihaploids. A haploid regenerant plant was sequenced (8× genome coverage) using Illumina technology. Genome data are disposable in the EMBL database as a standard for full genome and X chromosome assembly in this model species. Homozygous dihaploids were back-crossed with males to yield a progeny useful for the study of the evolution of the Y chromosome.

Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome.

To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR–Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR–Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses. Transient assays and transgenic experiments demonstrate that sgRNA/Cas9 constructs targeting the bean yellow dwarf virus inhibit the accumulation of the virus and confer resistance in transgenic N. benthamiana plants.

Silene dioica is a dioecious plant of the family Caryophyllaceae. In the present study, we used Illumina sequencing technology (MiSeq) to sequence, de novo assembly and annotate the transcriptomes of male and female copper tolerant S. dioica individuals. We sequenced the normalized mRNA of roots, shoots, flower buds and flowers for each sex. Raw reads of the transcriptome assembly project for S. dioica male and female individual have been deposited in NCBI's Sequence Read Archive (SRA) database with the accession number SRP094611. The Trinity and Detonate program was used to de novo assembly 92,347 transcripts for male and 94,757 transcripts for female transcriptome. The assembled transcriptome sequences for S. dioica male and female individuals can be accessed at NCBI with the following accession numbers: GFCG00000000 (male); GFCH00000000 (female). The obtained transcriptomic data will be useful for further studies focusing on copper tolerance, comparative transcriptome analysis with other Silene species and sex chromosomes evolution.

In contrast to animals, separate sexes and sex chromosomes in plants are very rare. Although the evolution of sex chromosomes has been the subject of numerous studies, the impact of repetitive sequences on sex chromosome architecture is not fully understood. New genomic approaches shed light on the role of satellites and transposable elements in the process of Y chromosome evolution. We discuss the impact of repetitive sequences on the structure and dynamics of sex chromosomes with specific focus on Rumex acetosa and Silene latifolia. Recent papers showed that both the expansion and shrinkage of the Y chromosome is influenced by sex-specific regulation of repetitive DNA spread. We present a view that the dynamics of Y chromosome formation is an interplay of genetic and epigenetic processes.

Silene latifolia (or white campion) possesses a well-established sex determination system with a dominant Y chromosome in males (the mammalian type). The heteromorphic sex chromosomes X and Y in S. latifolia largely stopped recombination; thus, we can expect a gradual genetic degeneration of the Y chromosome. It is well proven that neither diploid nor polyploid S. latifolia sporophytes can survive without at least one X, so the only life stage possessing the Y as the sole sex chromosome is the male gametophyte (pollen tube), while the female gametophyte seems to be X-dependent. Previous studies on anther-derived plants of this species showed that the obtained plants (largely haploid or dihaploid) were phenotypically and cytologically female. In this paper, we provide molecular evidence for the inviability of plants lacking the X chromosome. Using sex-specific PCR primers, we show that all plantlets and plants derived from anther cultures are female. In studying anther-derived diploid females by sequencing of X-linked markers, we demonstrate that these plants are really homozygous dihaploids. A haploid regenerant plant was sequenced (8× genome coverage) using Illumina technology. Genome data are disposable in the EMBL database as a standard for full genome and X chromosome assembly in this model species. Homozygous dihaploids were back-crossed with males to yield a progeny useful for the study of the evolution of the Y chromosome.

Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome.

Background Genome size evolution is a complex process influenced by polyploidization, satellite DNA accumulation, and expansion of retroelements. How this process could be affected by different reproductive strategies is still poorly understood. Methodology/Principal Findings We analyzed differences in the number and distribution of major repetitive DNA elements in two closely related species, Silene latifolia and S. vulgaris. Both species are diploid and possess the same chromosome number (2n = 24), but differ in their genome size and mode of reproduction. The dioecious S. latifolia (1C = 2.70 pg DNA) possesses sex chromosomes and its genome is 2.5× larger than that of the gynodioecious S. vulgaris (1C = 1.13 pg DNA), which does not possess sex chromosomes. We discovered that the genome of S. latifolia is larger mainly due to the expansion of Ogre retrotransposons. Surprisingly, the centromeric STAR-C and TR1 tandem repeats were found to be more abundant in S. vulgaris, the species with the smaller genome. We further examined the distribution of major repetitive sequences in related species in the Caryophyllaceae family. The results of FISH (fluorescence in situ hybridization) on mitotic chromosomes with the Retand element indicate that large rearrangements occurred during the evolution of the Caryophyllaceae family. Conclusions/Significance Our data demonstrate that the evolution of genome size in the genus Silene is accompanied by the expansion of different repetitive elements with specific patterns in the dioecious species possessing the sex chromosomes., PLoS ONE, 7 (2), ISSN:1932-6203

Background The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. Results Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences. Conclusions High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.

Background The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome. Results In the present study we provide evidence for the existence of an X linked copy of the AP3 gene. We further show that the Y copy is probably located in a chromosomal region where recombination restriction occurred during the first steps of sex chromosome evolution. A comparison of X and Y copies did not reveal any clear signs of degenerative processes in exon regions. Instead, both X and Y copies show evidence for relaxed selection compared to the autosomal orthologues in S. vulgaris and S. conica. We further found that promoter sequences differ significantly. Comparison of the genic region of AP3 between the X and Y alleles and the corresponding autosomal copies in the gynodioecious species S. vulgaris revealed a massive accumulation of retrotransposons within one intron of the Y copy of AP3. Analysis of the genomic distribution of these repetitive elements does not indicate that these elements played an important role in the size increase characteristic of the Y chromosome. However, in silico expression analysis shows biased expression of individual domains of the identified retroelements in male plants. Conclusions We characterized the structure and evolution of AP3, a sex linked gene with copies on the X and Y chromosomes in the dioecious plant S. latifolia. These copies showed complementary expression patterns and relaxed evolution at protein level compared to autosomal orthologues, which suggests subfunctionalization. One intron of the Y-linked allele was invaded by retrotransposons that display sex-specific expression patterns that are similar to the expression pattern of the corresponding allele, which suggests that these transposable elements may have influenced evolution of expression patterns of the Y copy. These data could help researchers decipher the role of transposable elements in degenerative processes during sex chromosome evolution.