Sexy ways: the methodical approaches to study plant sex chromosomes.

Sex chromosomes have evolved in many plant species with separate sexes. Current plant research is shifting from examining the structure of sex chromosomes to exploring their functional aspects. New studies are progressively unveiling the specific genetic and epigenetic mechanisms responsible for shaping distinct sexes in plants. While the fundamental methods of molecular biology and genomics are generally employed for the analysis of sex chromosomes, it is often necessary to modify classical procedures not only to simplify and expedite analyses but sometimes to make them possible at all. In this review, we demonstrate how, at the level of structural and functional genetics, cytogenetics, and bioinformatics, it is essential to adapt established procedures for sex chromosome analysis.

A Panicum-derived chromosomal segment captured by Hordeum a few million years ago preserves a set of stress-related genes.

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.

Chemical genetics in Silene latifolia elucidate regulatory pathways involved in gynoecium development.

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. In Silene 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. Eight candidates were found to have a positive role in gynoecium promotion, floral organ size, and whorl boundary, and affect the expression of class B MADS-box flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using field emission environmental scanning electron microscopy, and examined the differences between females and androhermaphrodites in their placenta and ovule organization. Our results reveal the regulatory pathways potentially involved in sex-specific flower development in the classical model of dioecy, S. latifolia. These pathways include previously hypothesized and unknown female-regulator genes that act on the factors that determine the flower boundaries, and a negative regulator of anther development, SUPERMAN-like (SlSUP).

Fundamentally different repetitive element composition of sex chromosomes in Rumex acetosa.

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.

Comparison of various approaches to detect algal culture contamination: a case study of Chlorella sp. contamination in a Phaeodactylum tricornutum culture.

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 <1:105. Our results confirmed PCR/qPCR to provide the most reliable and sensitive results, with detection sensitivity close to 75 cells/mL. The method was similarly sensitive in a pure C. vulgaris culture as well as in a mixed culture containing 107-times more P. tricornutum cells. A next-generation sequencing analysis revealed a positive discrimination of C. vulgaris during DNA extraction. The method of cultivation media exchange from sea water to fresh water, preferred by the Chlorella contaminant, demonstrated a presence of the contaminant with a sensitivity comparable to PCR approaches, albeit with a much longer detection time. The results suggest that a qPCR/PCR-based approach is the best choice for an early warning in the commercial culturing of microalgae. This method can be conveniently complemented with the substitution-cultivation method to test the proliferating potential of the contaminant. KEY POINTS: • PCR-based protocol developed for detection of Chlorella cells. • Synergy of various approaches shows deeper insight into a presence of contaminants. • Positive/negative discrimination occurs during DNA extraction in mixed cultures. • Newly developed assays ready to use as in diagnostics of contamination.

In-Depth Sequence Analysis of Bread Wheat VRN1 Genes.

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.

Identification of polycomb repressive complex 1 and 2 core components in hexaploid bread wheat.

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.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Sex and the flower - developmental aspects of sex chromosome evolution.

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.

Impact of repetitive DNA on sex chromosome evolution in plants.

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.

The X chromosome is necessary for somatic development in the dioecious Silene latifolia: cytogenetic and molecular evidence and sequencing of a haploid genome.

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.

Identification of a novel retrotransposon with sex chromosome-specific distribution in Silene latifolia.

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.

Possible mechanisms responsible for absence of a retrotransposon family on a plant Y chromosome.

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.

Omics-based analysis of honey bee (Apis mellifera) response to Varroa sp. parasitisation and associated factors reveals changes impairing winter bee generation.

The extensive annual loss of honey bees (Apis mellifera L.) represents a global problem affecting agriculture and biodiversity. The parasitic mite Varroa destructor, associated with viral co-infections, plays a key role in this loss. Despite years of intensive research, the complex mechanisms of Varroa - honey bee interaction are still not fully defined. Therefore, this study employed a unique combination of transcriptomic, proteomic, metabolomic, and functional analyses to reveal new details about the effect of Varroa mites and naturally associated factors, including viruses, on honey bees. We focused on the differences between Varroa parasitised and unparasitised ten-day-old worker bees collected before overwintering from the same set of colonies reared without anti-mite treatment. Supplementary comparison to honey bees collected from colonies with standard anti-Varroa treatment can provide further insights into the effect of a pyrethroid flumethrin. Analysis of the honey bees exposed to mite parasitisation revealed alterations in the transcriptome and proteome related to immunity, oxidative stress, olfactory recognition, metabolism of sphingolipids, and RNA regulatory mechanisms. The immune response and sphingolipid metabolism were strongly activated, whereas olfactory recognition and oxidative stress pathways were inhibited in Varroa parasitised honey bees compared to unparasitised ones. Moreover, metabolomic analysis confirmed the depletion of nutrients and energy stores, resulting in a generally disrupted metabolism in the parasitised workers. The combined omics-based analysis conducted on strictly parasitised bees revealed the key molecular components and mechanisms underlying the detrimental effects of Varroa sp. and its associated pathogens. This study provides the theoretical basis and interlinked datasets for further research on honey bee response to biological threats and the development of efficient control strategies against Varroa mites.

A new wild emmer wheat panel allows to map new loci associated with resistance to stem rust at seedling stage.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major wheat disease worldwide. A collection of 283 wild emmer wheat [Triticum turgidum L. subsp. dicoccoides (Körn. ex Asch. & Graebn.) Thell] accessions, representative of the entire Fertile Crescent region where wild emmer naturally occurs, was assembled, genotyped, and characterized for population structure, genetic diversity, and rate of linkage disequilibrium (LD) decay. Then, the collection was employed for mapping Pgt resistance genes, as a proof of concept of the effectiveness of genome-wide association studies in wild emmer. The collection was evaluated in controlled conditions for reaction to six common Pgt pathotypes (TPMKC, TTTTF, JRCQC, TRTTF, TTKSK/Ug99, and TKTTF). Most resistant accessions originated from the Southern Levant wild emmer lineage, with some showing a resistance reaction toward three to six tested races. Association analysis was conducted considering a 12K polymorphic single-nucleotide polymorphisms dataset, kinship relatedness between accessions, and population structure. Eleven significant marker-trait associations (MTA) were identified across the genome, which explained from 17% to up to 49% of phenotypic variance with an average 1.5 additive effect (based on the 1-9 scoring scale). The identified loci were either effective against single or multiple races. Some MTAs colocalized with known Pgt resistance genes, while others represent novel resistance loci useful for durum and bread wheat prebreeding. Candidate genes with an annotated function related to plant response to pathogens were identified at the regions linked to the resistance and defined according to the estimated small LD (about 126 kb), as typical of wild species.

Comparative analysis of a plant pseudoautosomal region (PAR) in Silene latifolia with the corresponding S. vulgaris autosome.

BACKGROUND: The sex chromosomes of Silene latifolia are heteromorphic as in mammals, with females being homogametic (XX) and males heterogametic (XY). While recombination occurs along the entire X chromosome in females, recombination between the X and Y chromosomes in males is restricted to the pseudoautosomal region (PAR). In the few mammals so far studied, PARs are often characterized by elevated recombination and mutation rates and high GC content compared with the rest of the genome. However, PARs have not been studied in plants until now. In this paper we report the construction of a BAC library for S. latifolia and the first analysis of a > 100 kb fragment of a S. latifolia PAR that we compare to the homologous autosomal region in the closely related gynodioecious species S. vulgaris. RESULTS: Six new sex-linked genes were identified in the S. latifolia PAR, together with numerous transposable elements. The same genes were found on the S. vulgaris autosomal segment, with no enlargement of the predicted coding sequences in S. latifolia. Intergenic regions were on average 1.6 times longer in S. latifolia than in S. vulgaris, mainly as a consequence of the insertion of transposable elements. The GC content did not differ significantly between the PAR region in S. latifolia and the corresponding autosomal region in S. vulgaris. CONCLUSIONS: Our results demonstrate the usefulness of the BAC library developed here for the analysis of plant sex chromosomes and indicate that the PAR in the evolutionarily young S. latifolia sex chromosomes has diverged from the corresponding autosomal region in the gynodioecious S. vulgaris mainly with respect to the insertion of transposable elements. Gene order between the PAR and autosomal region investigated is conserved, and the PAR does not have the high GC content observed in evolutionarily much older mammalian sex chromosomes.

Wild emmer wheat, the progenitor of modern bread wheat, exhibits great diversity in the VERNALIZATION1 gene.

Wild emmer wheat is an excellent reservoir of genetic variability that can be utilized to improve cultivated wheat to address the challenges of the expanding world population and climate change. Bearing this in mind, we have collected a panel of 263 wild emmer wheat (WEW) genotypes across the Fertile Crescent. The genotypes were grown in different locations and phenotyped for heading date. Genome-wide association mapping (GWAS) was carried out, and 16 SNPs were associated with the heading date. As the flowering time is controlled by photoperiod and vernalization, we sequenced the VRN1 gene, the most important of the vernalization response genes, to discover new alleles. Unlike most earlier attempts, which characterized known VRN1 alleles according to a partial promoter or intron sequences, we obtained full-length sequences of VRN-A1 and VRN-B1 genes in a panel of 95 wild emmer wheat from the Fertile Crescent and uncovered a significant sequence variation. Phylogenetic analysis of VRN-A1 and VRN-B1 haplotypes revealed their evolutionary relationships and geographic distribution in the Fertile Crescent region. The newly described alleles represent an attractive resource for durum and bread wheat improvement programs.

Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia.

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.

The Formation of Sex Chromosomes in Silene latifolia and S. dioica Was Accompanied by Multiple Chromosomal Rearrangements.

The genus Silene includes a plethora of dioecious and gynodioecious species. Two species, Silene latifolia (white campion) and Silene dioica (red campion), are dioecious plants, having heteromorphic sex chromosomes with an XX/XY sex determination system. The X and Y chromosomes differ mainly in size, DNA content and posttranslational histone modifications. Although it is generally assumed that the sex chromosomes evolved from a single pair of autosomes, it is difficult to distinguish the ancestral pair of chromosomes in related gynodioecious and hermaphroditic plants. We designed an oligo painting probe enriched for X-linked scaffolds from currently available genomic data and used this probe on metaphase chromosomes of S. latifolia (2n = 24, XY), S. dioica (2n = 24, XY), and two gynodioecious species, S. vulgaris (2n = 24) and S. maritima (2n = 24). The X chromosome-specific oligo probe produces a signal specifically on the X and Y chromosomes in S. latifolia and S. dioica, mainly in the subtelomeric regions. Surprisingly, in S. vulgaris and S. maritima, the probe hybridized to three pairs of autosomes labeling their p-arms. This distribution suggests that sex chromosome evolution was accompanied by extensive chromosomal rearrangements in studied dioecious plants.

Catalogue of stage-specific transcripts in Ixodes ricinus and their potential functions during the tick life-cycle.

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.