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.

• Keywords
• Bioinformatics, chromosome dissection, cytogenetics, dioecious plants, epigenetics, functional genetics, sex chromosomes, tandem repeats, transposable elements,
• MeSH
• Chromosomes, Plant * genetics   MeSH
• Sex Chromosomes * genetics   MeSH
• Plants genetics   MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

INTRODUCTION: Plants undergo various natural changes that dramatically modify their genomes. One is polyploidization and the second is hybridization. Both are regarded as key factors in plant evolution and result in phenotypic differences in different plant organs. In Silene, we can find both examples in nature, and this genus has a seed shape diversity that has long been recognized as a valuable source of information for infrageneric classification. METHODS: Morphometric analysis is a statistical study of shape and size and their covariations with other variables. Traditionally, seed shape description was limited to an approximate comparison with geometric figures (rounded, globular, reniform, or heart-shaped). Seed shape quantification has been based on direct measurements, such as area, perimeter, length, and width, narrowing statistical analysis. We used seed images and processed them to obtain silhouettes. We performed geometric morphometric analyses, such as similarity to geometric models and elliptic Fourier analysis, to study the hybrid offspring of S. latifolia and S. dioica. RESULTS: We generated synthetic tetraploids of Silene latifolia and performed controlled crosses between diploid S. latifolia and Silene dioica to analyze seed morphology. After imaging capture and post-processing, statistical analysis revealed differences in seed size, but not in shape, between S. latifolia diploids and tetraploids, as well as some differences in shape among the parentals and hybrids. A detailed inspection using fluorescence microscopy allowed for the identification of shape differences in the cells of the seed coat. In the case of hybrids, differences were found in circularity and solidity. Overal seed shape is maternally regulated for both species, whereas cell shape cannot be associated with any of the sexes. DISCUSSION: Our results provide additional tools useful for the combination of morphology with genetics, ecology or taxonomy. Seed shape is a robust indicator that can be used as a complementary tool for the genetic and phylogenetic analyses of Silene hybrid populations.

• Keywords
• Morphometrics geometrics, Silene dioica, Silene latifolia, elliptical Fourier analysis, plant hybrid, polyploidy, seed shape, symmetry,
• Publication type
• Journal Article MeSH

BACKGROUND: Sex chromosomes present a genomic region which to some extent, differs between the genders of a single species. Reliable high-throughput methods for detection of sex chromosomes specific markers are needed, especially in species where genome information is limited. Next generation sequencing (NGS) opens the door for identification of unique sequences or searching for nucleotide polymorphisms between datasets. A combination of classical genetic segregation analysis along with RNA-Seq data can present an ideal tool to map and identify sex chromosome-specific expressed markers. To address this challenge, we established genetic cross of dioecious plant Rumex acetosa and generated RNA-Seq data from both parental generation and male and female offspring. RESULTS: We present a pipeline for detection of sex linked genes based on nucleotide polymorphism analysis. In our approach, tracking of nucleotide polymorphisms is carried out using a cross of preferably distant populations. For this reason, only 4 datasets are needed - reads from high-throughput sequencing platforms for parent generation (mother and father) and F1 generation (male and female progeny). Our pipeline uses custom scripts together with external assembly, mapping and variant calling software. Given the resource-intensive nature of the computation, servers with high capacity are a requirement. Therefore, in order to keep this pipeline easily accessible and reproducible, we implemented it in Galaxy - an open, web-based platform for data-intensive biomedical research. Our tools are present in the Galaxy Tool Shed, from which they can be installed to any local Galaxy instance. As an output of the pipeline, user gets a FASTA file with candidate transcriptionally active sex-linked genes, sorted by their relevance. At the same time, a BAM file with identified genes and alignment of reads is also provided. Thus, polymorphisms following segregation pattern can be easily visualized, which significantly enhances primer design and subsequent steps of wet-lab verification. CONCLUSIONS: Our pipeline presents a simple and freely accessible software tool for identification of sex chromosome linked genes in species without an existing reference genome. Based on combination of genetic crosses and RNA-Seq data, we have designed a high-throughput, cost-effective approach for a broad community of scientists focused on sex chromosome structure and evolution.

• MeSH
• Genetic Markers genetics   MeSH
• Genome, Human MeSH
• Genes, X-Linked * MeSH
• Genes, Y-Linked * MeSH
• Polymorphism, Single Nucleotide genetics   MeSH
• Humans MeSH
• Polymerase Chain Reaction MeSH
• RNA genetics   MeSH
• Sequence Analysis, RNA methods   MeSH
• Software * MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Genetic Markers MeSH
• RNA MeSH

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.

• MeSH
• Alleles MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• Exons MeSH
• Introns MeSH
• Evolution, Molecular * MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Plant MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Retroelements MeSH
• Genes, Plant MeSH
• Plant Proteins genetics   MeSH
• Sequence Analysis, DNA MeSH
• Silene genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Retroelements MeSH
• Plant Proteins MeSH

Understanding the origin and evolution of sex chromosomes requires studying recently evolved X-Y chromosome systems such as those in some flowering plants. We describe Y chromosome deletion mutants of Silene latifolia, a dioecious plant with heteromorphic sex chromosomes. The combination of results from new and previously described deletions with histological descriptions of their stamen development defects indicates the presence of two distinct Y regions containing loci with indispensable roles in male reproduction. We determined their positions relative to the two main sex determination functions (female suppressing and the other male promoting). A region proximal to the centromere on the Y p arm containing the putative stamen promoting sex determination locus includes additional early stamen developmental factors. A medial region of the Y q arm carries late pollen fertility factors. Cytological analysis of meiotic X-Y pairing in one of the male-sterile mutants indicates that the Y carries sequences or functions specifically affecting sex chromosome pairing.

• MeSH
• Biological Evolution * MeSH
• DNA, Plant genetics   MeSH
• Species Specificity MeSH
• Genetic Variation MeSH
• Mutation MeSH
• Sex Chromosomes genetics   MeSH
• Recombination, Genetic * MeSH
• Genes, Plant genetics   MeSH
• Reproduction genetics   MeSH
• Selection, Genetic MeSH
• Silene genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH